Methods of screening using amphibians

ABSTRACT

High-throughput methods of screening agents for activities affecting renal, cardiac, blood or lymphatic vascular development and functions in amphibians in multiwell plates are provided. Also provided are novel compounds that modulate blood and lymphatic vascular development.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/161,497, filed Mar. 19, 2009, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides for in vivo chemical screening methodsinvolving a simple phenotypic read-out (edema formation or lethality),optionally followed by in situ hybridization or immunohistochemistry toscreen for agents modulating cardiac, vascular, lymphatic, or renaldevelopment or organ functions in amphibian embryos or tadpoles in amultiwell format. Furthermore, active compounds interfering with bloodvascular and lymphatic development in Xenopus laevis are disclosed. Alsoprovided are screening methods to identify pathways that mediatelymphatic and/or vascular development in an amphibian.

BACKGROUND OF THE INVENTION

Lymphatic vessels play a major role in tissue pressure homeostasis,immune responses, and the uptake of dietary fat and fat-solublevitamins, as well as in inflammation and cancer progression (Cueni andDetmar, 2006). Recent studies indicate that both lymphatic and bloodvessels are involved in chronic inflammatory diseases such as rheumatoidarthritis, inflammatory bowel disease and psoriasis (Alitalo et al.,2005; Carmeliet, 2003; Cueni and Detmar, 2006). But the formation andactivation of both types of endothelium have also important roles in theprogression and metastasis of the majority of human cancers (Alitalo etal., 2005; Carmeliet, 2003). Tumors need to induce the growth of newblood vessels (angiogenesis) in order to secure the sufficient supply ofoxygen and nutrients. The growth of new lymphatic vessels(lymphangiogenesis) has been shown to promote cancer metastasis tosentinel lymph nodes and beyond (Hirakawa et al., 2007; Hirakawa et al.,2005; Mandriota et al., 2001; Skobe et al., 2001; Stacker et al., 2001),a phenomenon which is also found in human neoplasm (Dadras et al. 2005;Tobler and Detmar, 2006). Indeed, studies have revealed thattumor-induced lymphangiogenesis around the primary neoplasm is the mostsignificant prognostic indicator to predict the occurrence of regionallymph node metastasis in human malignant melanomas of the skin (Dadraset al., 2005). More recently, it has been found that tumors can inducelymphangiogenesis in their draining lymph nodes, even before theymetastasize and that induction of lymph node lymphangiogenesis promotesthe further metastatic cancer spread to distant sites (Hirakawa et al.,2007; Hirakawa et al., 2005). Thus, tumor-induced lymphatic growth andactivation represents a promising target for treating or preventingadvanced cancer. As a result, there has been a surge of interest inidentifying key players that can be used to specifically target theseprocesses therapeutically.

A strong correlation between the expression levels of thelymphangiogenic factor vascular endothelial growth factor-C (VEGFC),tumor lymphangiogenesis and lymph node metastasis has been found inhuman and in experimental tumors (Pepper et al., 2003). VEGFC promoteslymphangiogenesis by activating VEGF receptor-2 (VEGFR2) and VEGFR3 onlymphatic endothelial cells (Makinen et al., 2001). VEGF-C-deficientmice fail to develop a functional lymphatic system (Karkkainen et al.,2004), and transgenic expression of a soluble VEGFR-3 results inpronounced lymphedema (Makinen et al., 2001). However, blockade of theVEGF-C/VEGFR-3 axis only partially inhibits lymphatic metastasis,indicating that additional pathways are involved in mediating theformation and growth of lymphatic vessels. There have been previousattempts to identify lymphatic specific receptors and pathways bytranscriptional and proteomic profiling of cultured lymphaticendothelial cells (LEC) (Hirakawa et al., 2003; Petrova et al., 2002;Roesli et al., 2008). However, large-scale functional in vivo screens toidentify molecular pathways or drug-like small molecule modulators oflymphatic vessel formation have been missing to date.

In the last years, cost-efficient maintenance together with abundantexperimental techniques and molecular tools, have made zebrafish theonly vertebrate model used for large-scale in vivo drug screens (Zan andPeterson, 2005). Amphibians offer many of the same experimentaladvantages that have favored zebrafish in the past, such as rapidextra-uterine development, the transparency of developing tadpoles, andthe permeability of the skin for small molecules, but they have to datenot been employed for large-scale chemical library screens to gaininsight into vascular development. Amphibians have a common evolutionaryhistory with mammals that is an estimated 100 million years longer thanbetween zebrafish and mammals (Brändli, 2004). Being both tetrapods,amphibians and mammals share extensive synteny at the level of thegenomes and have many similarities in organ development, anatomy, andphysiology (Christensen et al., 2008; Raciti et al., 2008). These traitsfavor the use of amphibians for large-scale in vivo drug screens. In thepast, embryos and tadpoles of the African clawed frog (Xenopus laevis)have served as a powerful animal model to study blood vasculardevelopment and angiogenesis (Cleaver and Krieg. 1998; Helbling et al.,2000; Kälin et al., 2007: Levine et al., 2003). More recently, Xenopusembryos were shown to develop also a complex, well-defined lymphaticvascular system (Ny et al., 2005). Similar to the development of themammalian lymphatic vascular system, LECs transdifferentiate from venousblood vascular endothelial cells (BVEC) and lymphangioblasts contributein Xenopus to newly forming lymph vessels that mature to drain fluidsfrom the peripheral tissues back to the blood circulation.Antisense-morpholino knockdown studies of the lymphangiogenic factorVEGFC in Xenopus embryos causes lymphatic vessel defects similar to thephenotype observed in VEGFC-deficient mice, including impaired LECsprouting and migration, and the formation of lymphedema (Karkkainen etal., 2004: Ny et al. 2005).

Various publications have described the use of Xenopus embryos in thestudy of angiogenesis and lymphangiogenesis. For example, U.S. PatentApplication Publication No. 2006/0159676 A1 describes methods ofinhibiting and promoting various physiological processes, includingangiogenesis and lymphangiogenesis by interference with the apelin/APJsignaling pathway, as well as methods of identifying therapeutic agentsaffecting the apelin/APJ signaling pathway. The reference describes theeffects of various treatments on apelin expression in frog embryos, asmeasured by in situ hybridization, but does not describe an anatomicalpattern of edema formation.

U.S. Patent Application Publication No. 2007/0107072 describestransgenic amphibian models for lymphatic vessel development, includingassays that allow screening for compounds able to modulatelymphangiogenesis. The reference describes the use of transgenic frogembryos to study the development of the lymphatic vascular network. Thereference does not, however, describe an anatomical pattern of edemaformation.

There is, therefore, a need for additional methods for in vivo screeningin amphibian model systems and for compounds identified using suchscreens.

SUMMARY OF THE INVENTION

The present invention addresses these problems by providing novelmethods for in vivo screening and compounds identified using suchmethods.

In one aspect, the invention provides a method of screening, comprisingthe steps of: a) treating a plurality of amphibians with a plurality ofagents; b) identifying an amphibian from the plurality of amphibianswherein the treatment causes edema in or death of the amphibian: and c)determining the anatomical pattern of edema formation in the identifiedamphibian.

In some embodiments, the plurality of amphibians are treated byincluding the plurality of agents in the culture media containing theplurality of amphibians.

In some embodiments, the plurality of agents are dissolved in theculture media containing the plurality of amphibians.

In some embodiments, the method is performed in a multi-well format.

In some embodiments, the plurality of amphibians are a plurality ofembryos, tadpoles, or adults.

In some embodiments, the plurality of amphibians are from the subclassLissamphibia.

In more specific embodiments, the plurality of amphibians are frogs,toads, newts, salamanders, mudpuppies, or caecilians.

In some embodiments, the plurality of amphibians are from the genusXenopus.

In more specific embodiments, the plurality of amphibians are from thespecies Xenopus laevis or Xenopus tropicalis.

In some embodiments, the plurality of agents are independently smallmolecules, drugs, antibodies, peptides, secreted proteins, nucleicacids, antisense RNA molecules, ribozymes, RNA interference nucleotidesequences, antisense oligomers, or morpholino oligonucleotides.

In some embodiments, the edema or death is caused by an activity in thevascular, lymphatic, cardiac, or excretory system of the identifiedamphibian.

In some embodiments, the method further comprises the step ofidentifying the target tissue or organ of the agent responsible for theedema or death in the identified amphibian.

In other embodiments, the anatomical pattern of edema formation in theidentified amphibian is cerebral, periocular, pericardial, ventral,proctodeal, pronephric, or tail tip.

In still other embodiments, the anatomical pattern of edema formation inthe identified amphibian is a cardiac phenotype or a lymph-heartenlargement.

In some embodiments, the step to identify the amphibian is performed bya secondary screen.

In more specific embodiments, the secondary screen is performed by insitu hybridization or by immunohistochemistry.

In other more specific embodiments, the in situ hybridization isperformed manually, semi-automated or fully automated.

In some embodiments, the method further comprises the step of d)identifying the agent causing the edema in or death of the amphibian.

According to another aspect, the invention provides a compoundidentified by an in vivo screening method comprising the steps of: a)treating a plurality of amphibians with a plurality of agents; b)identifying an amphibian from the plurality of amphibians wherein thetreatment causes edema in or death of the amphibian; c) determining theanatomical pattern of edema formation in the identified amphibian; andd) identifying the agent causing the edema in or death of the amphibian.

In more specific embodiments, the methods used to identify the compoundare as described above.

In some embodiments of the invention, the compound affects blood vesseldevelopment only.

In more specific embodiments of the invention, the compound causesdefective vasculogenesis.

In other more specific embodiments, the compound causes defectiveangiogenesis.

In still other more specific embodiments, the compound causes ectopicangiogenic sprouting.

In still other more specific embodiments, the compound causes vitellinevein network hypoplasia.

In some embodiments of the invention, the compound affects blood andlymph vessel formation.

In more specific embodiments of the invention, the compound causesdefective blood and lymph angiogenesis.

In other more specific embodiments, the compound causes vitelline veinnetwork hyperplasia and defective lymph angiogenesis.

In some embodiments of the invention, the compound affects lymph vesselformation only.

In some embodiments of the invention, the compound causes defectivelymph angiogenesis.

In another aspect, the invention provides a method for in vivo screeningcomprising: a) treating a plurality of amphibians with a plurality ofagents; b) identifying an amphibian from the plurality of amphibianswherein the treatment causes edema in or death of the amphibian; c)determining the anatomical pattern of edema formation in the identifiedamphibian: and d) identifying a pathway that mediates lymphatic and/orvascular development in the identified amphibian.

In some embodiments of the invention, the pathway is a VEGF pathway.

In some embodiments of the invention, the pathway is targeted by anadenosine receptor antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A chemical library screening method to identify small-moleculemodulators of vascular development in Xenopus embryos. Xenopus embryoswere arrayed in multi-well plates and single compounds from theLOPAC¹²⁸⁰ chemical library were added to the water in each well. Embryoswere screened visually for developmental defects (edema and/or latelethality). Positive hits are verified by repeating the phenotypicassay. Compounds interfering with vascular development are identified bywhole mount in situ hybridization of compound-treated Xenopus embryos.Note the screen can also recover compounds affecting cardiac or renaldevelopment or function.

FIG. 2. Distinct edema phenotypes induced by small-molecule compounds inXenopus tadpoles. A selection of compound-treated Xenopus tadpolesdisplaying different classes of edema morphologies is shown (see Table 8for details): class A (a-f, r); class B (g, h), class C (i, j), class D(k, l), class E (m, n), and class F (o-q). Pictures of compound-treated(a-r) and control DMSO-treated embryos (s, t) were taken during thescreening process at the indicated developmental stages. Xenopustadpoles are generally shown in lateral views. Arrowheads mark primaryregions of edema formation. Where required, dorsal views (h, l, t) areshown to highlight particular edema morphologies. Alternatively,magnifications of specific regions such as the head for periocular (b)and cerebral (n) edemas, heart for pericardial edemas (j, p), pronephrickidneys (d) and lymph hearts (f) are shown. Abbreviations: GBR-12909,GBR-12909 dihydrochloride; Phenanthridinone, 6(5H)-Phenanthridinone;Tyr, Tyrphostin.

FIG. 3. Compounds affecting distinct aspects of blood vessel developmentin vivo. Compound-treated (a-n) and control DMSO-treated Xenopus embryos(o, p) were analyzed by whole mount in situ hybridization for expressionof the vascular marker gene apj. Stage 35/36 embryos are shown inlateral views with anterior to the left. Close-up views of the trunkillustrate the morphology of the blood vessels. Compound names areindicated. (a-d) Hypoplastic VVN (asterisks) and PCV (arrowheads). (e,f) Lack of ISVs (arrowheads). Note that assembly of PCV and VVN(asterisk) is unaffected. (g, h) Ectopic ISV (arrowheads) and dysplasticVVN (asterisk), (i, j) Ectopic ISV (arrowheads) and hyperplastic VVN(asterisk). (k-n) Hypoplastic, dispersed VVN (asterisks), but normal ISVangiogenesis (arrowheads). (o, p) Control embryos with normal VVN(asterisk), PCV (arrow), and ISV (arrowhead). Abbreviations:Calmidazolium, calmidazolium chloride; Indirubin, indirubin-3′-oxime.

FIG. 4. Compounds affecting blood and lymph vessel development inXenopus tadpoles. Control DMSO-(a, b) and compound-treated (c-j) Xenopusembryos were analyzed by whole mount in situ hybridization forexpression of the blood vascular marker gene apj at stage 35/36 and thelymphatic marker gene vegfr3 at stage 42. PaneLs of the embryonic bloodvasculature (apj) are accompanied by close-up views illustrating ISVangiogenesis and VVN development in the embryonic trunk. The panelsvisualizing the developing lymphatic system (vegfr3) include close-upsof the head and midtrunk region (middle panels) for the anterior lymphsacs (ALS) and the anterior lymph hearts (ALH), and enlargements (leftpanels) of the tail for posterior lymph vesseLs (PLV). (a) Normal ISVs(arrowhead) and VVN (asterisk). (b) Normal ALS (arrow), ALH (asterisk),and PLV (arrowheads). (c) Stunted ISVs (arrowhead), normal VVN(asterisk). (d) Hypoplastic PLV (arrowheads), impaired ALS (arrow) andALH (asterisk) lymphatics. (e) Stunted ISV (arrowhead), hypoplastic VVN(asterisk). (f) Stunted ALS lymphatics (arrow), dysplastic ALHlymphatics (asterisks), hypoplastic PLY (arrowheads). (g) Normal ISV(arrowhead), hyperplastic VVN. (h) Hypoplastic PLV (arrowheads),impaired ALS (arrow) and ALH (asterisks) lymphatics. (i) Stunted ISV(arrowhead), hyperplastic VVN. (j) Complete lack of ALS (arrow), ALH(asterisk) and tail lymphatics. Note that vegfr3 expression persists inthe VVN (arrowhead). Abbreviations: 7-Cyclo,7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine;Naphthalimide, 4-amino-1,8-naphthalimide; Naphthyridine,7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine.

FIG. 5. Compounds specifically affecting lymph vessel development inXenopus tadpoles. Control DMSO-(a, b) and compound-treated (c-n) Xenopusembryos were analyzed by whole mount in situ hybridization as describedin the legend to FIG. 4. Close-up views of apj-stained embryosillustrate normal ISV angiogenesis and VVN development in the embryonictrunk. ALS, ALH, and PLVs are highlighted by arrows, asterisks, andarrowheads, respectively. (a, b) Normal ALS and ALH lymphatics, andPLVs. (c, d) Dysplastic ALH lymphatics. (e, f) Dysplastic ALS and ALHlymphatics, and hypoplastic PLVs. Note persistent vegfr3 expression inthe VVN (black arrowhead). (g, h) Stunted ALS lymphatics, dysplastic ALHlymphatics, and hypoplastic PLVs. (i, j) Impaired ALS lymphangiogenesis,stunted ALH lymphatics, and hypoplastic PLVs. (k-n) Impaired ALS and ALHlymphangiogenesis, and hypoplastic PLVs.

FIG. 6. Selective and cell-type specific in vitro responses ofendothelial cells to treatment with small-molecule compounds. 24compounds were tested in vitro on human lymphatic (LEC; black bars) andblood vascular (HUVEC; open bars) endothelial cell cultures for effectson cell proliferation and tube formation. (a) Results of the compoundscreens using cell proliferation assays. Compounds were screened at adose of 10 μM in 0.1% DMSO. Control cultures were treated with 0.1%DMSO. (b) Results of the compound screens using tube formation assays.Compounds were screened at a dose of 1 μM in 0.1% DMSO. Control cultureswere treated with 0.1% DMSO. Abbreviations: 7-Cyclo:7-cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine;L-687,384: L-687,384 hydrochloride; Naphthalimide:4-amino-1,8-naphthalimide; Naphthyridine:7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine; Nicardipine: Nicardipinehydrochloride; Phenylxanthine: 1,3-diethyl-8-phenylxanthine. Barsindicate mean values and standard deviations of three independentlyperformed assays.

FIG. 7. Differential, cell-type specific effects of selectedsmall-molecule compounds on endothelial tube formation. Confluentmonolayers of HUVEC or LEC were overlaid with collagen type I gelscontaining the indicated compounds at a dose of 1 μM in 0.1% DMSO. Scalebars: 100 μm.

FIG. 8. The adenosine A1 receptor antagonist naphthyridine inhibitsVEGFA-induced angiogenesis and lymphatic vessel enlargement in mice.VEGFA-containing Matrigel plugs were implanted subcutaneously into adultmice and the mice were subsequently treated systemically withnaphthyridine or with vehicle control for 6 days. (a-c) Differentialimmunofluorescence analysis for the lymphatic-specific marker LYVE1(green, examples highlighted by arrows) and the pan-vascular marker CD31(red) demonstrated lymphatic vessel enlargement and enhanced numbers ofblood vessels in the skin surrounding VEGFA-containing Matrigels (b), ascompared with Matrigels containing PBS (a). Treatment with naphthyridineresulted in a reduction of blood vessel numbers and lymphatic vesselenlargement (c). (d-g) Quantitative image analyses confirmed that thedensity of lymphatic vessels was unchanged by VEGFA alone or by combinedVEGFA and naphthyridine treatment (d). In contrast, the tissue areacovered by lymphatic vessels surrounding VEGFA containing Matrigels wassignificantly reduced by treatment with naphthyridine (e). Treatmentwith naphthyridine also reduced the number of VEGFA-induced bloodvessels (f) and the tissue area covered by blood vessels (g). Scalebars: 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis and lymphangiogenesis are essential for organogenesis, butalso play important roles in tissue regeneration, chronic inflammation,and tumor progression. Furthermore, defects in the development orfunction of the cardiovascular and excretory systems underlie majordiseases in humans, such as myocardial infarction, stroke, hypertension,and chronic kidney disease. Provided herein are in vivo methods toscreen a plurality of agents, such as, for example a chemical library,to identify novel agents and biological mechanisms affecting cardiac,lymphatic, vascular, or renal function in amphibian embryos andtadpoles. In one embodiment of the invention, a novel screening methodinvolving a simple phenotypic read-out (edema formation or lethality)followed by in situ hybridization is used to screen an annotatedchemical library of 1,280 bioactive compounds in an multiwell format. Inalternative embodiments of the invention, the in situ hybridization stepis replaced by immunohistochemical techniques using specific antibodies.Using a two-step screening method of the instant invention, compoundsinterfering with blood vascular and/or lymphatic development in Xenopuslaevis are identified. The compounds identified according to the instantscreening methods may be used directly in preclinical models ofinflammation and cancer metastasis.

The instant invention provides in one aspect an unbiased chemicalscreening approach in combination with a simple phenotypic readout andin situ hybridization (manual, semi-automated, or fully automated) toidentify agents and pathways involved in the development of the cardiac,renal, lymphatic and blood vascular system in Xenopus tadpoles.

Also provided according to another aspect of the invention are compoundsidentified according to the methods of the instant chemical screen.Compounds identified in the screen include compounds affecting vascularand lymphatic development.

In yet another aspect of the invention, novel pathways and targets areidentified using the screening methods disclosed herein. Pathways notpreviously known to mediate lymphatic and/or vascular development arerevealed by the screening methods of the invention. In one embodiment,the screening method is used to identify an adenosine A1 receptorantagonist that inhibits lymphatic and blood vessel formation in Xenopustadpoles.

Many of the compounds identified in one of the in vivo screenembodiments of the invention are also active in at least one of fourdifferent endothelial in vitro assays, such as cell proliferation andtube formation. Taken together, the various aspects of the instantinvention establish rapid and sensitive in vivo methods for large-scalechemical screens using amphibians to identify novel pathways and leadcompounds with selectivity for lymphatic and blood vessel formation in atime- and cost-saving manner.

The disclosed screening methods also provide for the identification ofand recovery of compounds affecting cardiac and excretory systemdevelopment and function. The whole-organism based screening methods ofthe instant invention are more informative than in vitro screeningassays and therefore accelerate the development and testing of new drugcandidates for the treatment of disorders such as chronic inflammationand cancer.

In one aspect, the invention provides a method for in vivo screeningcomprising: a) treating a plurality of amphibians with a plurality ofagents; b) identifying an amphibian from the plurality of amphibianswherein the treatment causes edema in or death of the amphibian; and c)determining the anatomical pattern of edema formation in the identifiedamphibian.

According to some embodiments of the invention, the plurality of agentsused in the screening methods are small molecules, drugs, antibodies,peptides, secreted proteins, nucleic acids, antisense RNA molecules,ribozymes, RNA interference nucleotide sequences, antisense oligomers,or morpholino oligonucleotides. However, any agent having an effect ofinterest on the plurality of amphibians could potentially be of interestand use in carrying out the methods of the invention.

In preferred embodiments of the invention, the plurality of agents usedin the screening methods are part of a library of agents such as, forexample, a chemical or compound library. For purposes of the invention,a chemical library or compound library is a collection of storedchemicals (agent or compound) usually used in low- and high-throughputdrug discovery screening procedures. Typically, the chemical libraryconsists of a series of stored chemicals with a specific chemicalcomposition. Each chemical is associated with information stored in somekind of database with information such as the chemical structure,purity, quantity, and physiochemical characteristics of the chemical. Indrug discovery screening, such as, for example, in some of the screeningmethods of the instant invention, it is desirable to screen a drugtarget or a biological process against a selection of chemicals thatrepresent as much of the appropriate chemical space as possible. Theterm “drug target” describes the native protein or other biomolecule inthe body whose activity is affected by a drug (i.e. chemical, compound,or agent) resulting in a particular biological or therapeutic effect.The targets may include without limitation: G protein coupled receptors,enzymes (such as protein kinases), ligand-gated ion channels,voltage-gated ion channels, solute carriers, nuclear hormone receptors,structural proteins, nucleic acids (including e.g., deoxyribonucleicacids and ribonucleic acids), lipids, lipoproteins, and membranes.

The plurality of agents are used to treat a plurality of amphibiansaccording to the methods of the instant invention. As would beunderstood by those of ordinary skill in the art, the plurality ofagents may be usefully delivered to the plurality of amphibians by anymeans. For example, in some embodiments of the invention, the pluralityof amphibians are treated by including the plurality of agents in theculture media containing the amphibians. In preferred embodiments, theplurality of agents are dissolved in the culture media containing theplurality of amphibians.

It would also be understood by those of ordinary skill in the art thatthe plurality of amphibians are typically treated independently with theplurality of agents according to the methods of the invention. In otherwords, individual amphibians are separately treated with individualagents so that specific effects of each agent on an individual amphibiancan be determined. It is within the scope of the invention, however,that mixtures of agents could also be used to treat individualamphibians according to the instant methods. Such mixtures may be usefulin identifying agents having combinatorial activities on the amphibiansin the screening methods. It is also within the scope of the inventionthat other conditions, such as, e.g., concentration of agent,composition of culture media, temperature, and so on, may also be variedas desired in the practice of the instant methods.

In preferred embodiments of the invention, the plurality of agents aredissolved in the culture media containing the plurality of amphibians.However, other means of treatment of the amphibians by the agents areunderstood to be within the scope of the invention, as would be apparentto one of ordinary skill in the art.

In some embodiments of the invention, the methods are performed in amulti-well format. As is understood by those of ordinary skill in theart, multi-well format typically refers to a specific arrangement ofplastic Petri dishes (also known as culture plates), where two or moredishes are incorporated into one plastic lidded container to create whatis called a multi-well plate. Multi-well plates may typically harbor 6,12, 24, 48, 96 or 384 wells. The multiwell plate format is designedspecifically for high-throughput experiments on a small scale. In thecontext of the present invention, multi-well format may refer to the useof 12-, 24-, or 48-well plates to culture embryos or tadpoles for theparallel testing and characterization of thousands of chemical agents.

The methods of the instant invention include the step of identifying anamphibian from the plurality of amphibians, wherein the treatment causesedema in or death of the amphibian. In other words, the methods includea procedure or test to identify agents, which may be present in achemical library, that elicit a desired biological activity. The screenmay test agents on whole amphibian organisms to identify agents thatelicit a phenotype of interest. A phenotype is defined as any observablecharacteristic or trait of an organism: such as its morphology,development, biochemical, metabolic or physiological properties, orbehavior. The phenotypes are compared with control organisms, which aremock treated or are untreated. The observed phenotype is the result ofthe agent modulating the expression of the organism's genes, theactivity of its gene products (e.g. mRNA, proteins) or otherconstituents (e.g. lipids, metabolic products). Typically, the screen issimple, fast, and cost-effective. These characteristics permit the rapidselection of agents with desirable bioactivity.

In some embodiments of the invention, the plurality of amphibians are aplurality of embryos, tadpoles, or adults. As used herein, the term“embryo” is defined as a multicellular diploid eukaryote in its earlieststage of development, from the time of first cell division until birth,hatching or germination. The development of a fertilized egg (zygote)into embryo is called embryogenesis. In animals, the development of thezygote into an embryo proceeds through specific recognizable stages ofblastula, gastrula, neurula, and organogenesis. In amphibians,fertilized eggs and the resulting multicellular organisms are called byconvention embryos from fertilization through hatching, which is definedas the stage when the embryo breaks the covering vitelline membrane.After hatching, the free-swimming amphibian is called a tadpole.

As used herein, the term “tadpole” is defined as the wholly aquaticlarval stage in the life cycle of an amphibian, particularly of a frogor toad. Tadpoles are young amphibians that live in the water. Duringthe tadpole stage of the amphibian life cycle, most respire by means ofautonomous external or internal gills. Tadpoles do not usually havelimbs (arms or legs) until the transition to adulthood.

As used herein, the term “adult” is defined as a biologically grown ormature organism. In amphibians, a tadpole matures into an adult byundergoing metamorphosis, a biological process of transformation,involving a conspicuous and relatively abrupt change in the animal'sbody structure through cell growth and differentiation. The tadpolemetamorphosizes by gradually growing limbs (usually the legs first,followed by the arms) and then outwardly absorbing its tail by apotosis.Lungs develop around the time of leg development. During the finalstages of external metamorphosis, the tadpole's mouth changes from asmall, enclosed mouth at the front of the head to a large mouth the samewidth as the head. The intestines shorten to make way for the new diet.With the completion of metamorphosis an adult organism emerges, which isusually (but not always) accompanied by a change of habitat usually fromwater to land.

In some embodiments of the invention, the methods further comprise thestep of identifying the target tissue or organ of the agent responsiblefor the edema or death in the identified amphibian. As used herein, theterms target tissue or organ refer to cellular structures of theorganism (e.g. embryo, tadpole or adult) that are affected by thebiological or pharmacological activity of an agent, which had beenabsorbed by the organism. Tissue is in this context considered acellular organizational level intermediate between cells and a completeorgan. Hence, a tissue is an ensemble of cells, not necessarilyidentical, but from the same origin, that together carry out a specificfunction. Animal tissues are typically grouped into four basic types:connective tissue, muscle tissue, nervous tissue, and epithelial tissue.The functional grouping and assembly of multiple tissues then formorgans. An organ is therefore a collection of tissues joined in astructural unit to serve one or more common physiological functions.

The methods of the instant invention further include the step ofdetermining the anatomical pattern of edema formation in the identifiedamphibian. As described in detail in the Examples, edema formation inresponse to compound treatment in an amphibian may be initially highlyregionalized, i.e. restricted to a specific organ or tissue, beforebecoming generalized to the whole body. Hence, according to the methodsof the invention, the anatomical distribution and the temporal onset ofedemas resulting from the treatment step may be analyzed to determine ananatomical pattern.

In some embodiments of the invention, the anatomical pattern of edemaformation in an amphibian identified according to the method iscerebral, periocular, pericardial, ventral, proctodeal, pronephric, ortail tip. In some embodiments of the invention, the anatomical patternof edema formation is a cardiac phenotype or a lymph-heart enlargement.

In some embodiments of the instant invention, a secondary screen isimplemented on the basis of the results obtained in the initial orprimary screen. The purpose of this secondary screen is to identify andcharacterize in greater detail the biological activity of agentsrecovered in the primary screen. In preferred embodiments of theinvention, the secondary screen is performed by in situ hybridization orimmunohistochemical procedures. In each procedure, molecular markers(antigens or nucleic acids) are visualized, and changes in theirexpression are compared to control organisms, which are mock treated orare untreated. Common screenable phenotypes in the secondary screeninvolve, for example, absent, enhanced, abnormal, and ectopic expressionof the molecular marker gene monitored in the secondary screen. Changesin the expression of a marker gene may provide important clues aboutchanges in morphogenesis, tissue formation, organogenesis or homeostasisafter the organism was exposed to the agent.

In preferred embodiments of the invention, the step to identify theamphibian is performed by in situ hybridization. In situ hybridizationis a type of hybridization procedure that uses a labeled complementaryoligonucleotide, deoxyribonucleic acid (cDNA) or ribonucleic acid (RNA)strand typically termed the “probe” to localize a specific DNA or RNAsequence in a portion or section of a tissue in situ (i.e. in theplace). Alternatively, in situ hybridization can be carried out in wholemount on an entire organism, for example if the whole organism is smallenough (e.g. embryo, larvae, and tadpole). Hybridization is the processof establishing a non-covalent, sequence-specific interaction betweentwo or more complementary strands of nucleic acids into a single hybrid,which in the case of two strands is referred to as a duplex.Oligonucleotides, DNA, or RNA will bind to their complement under normalconditions, so that two perfectly complementary strands will bind toeach other readily. In situ hybridization is distinct fromimmunohistochemistry, which localizes proteins in tissue, organs, orwhole organisms.

In the context of the present invention, oligonucleotide, DNA, or RNAhybridization may be used in some embodiments to measure and localizemessenger RNA (mRNAs), microRNA (miRNA) and other gene transcripts incells of a tissue, organ, or intact organisms. In situ hybridizationsamples are whole organisms (e.g. embryos, tadpoles, larvae, adults),organs, or tissues or sections thereof. In situ hybridization may beused in the diagnosis of normal as well as abnormal cells such as thosefound in tissues and organs that have undergone pathological changes dueto cancer or other illnesses. The molecular markers (i.e., mRNAmolecules) detected by in situ hybridization may reflect the severity orpresence of some disease state. In situ hybridization may also be usedto understand the distribution and localization of biomarkers (i.e.differentially expressed mRNA molecules) in different parts of abiological sample. Specific biomarkers are characteristic of particularcellular events such as proliferation, differentiation, or cell death(apoptosis). A biomarker or molecular marker may in this context be, forexample, an mRNA molecule native to the tissue, organ, or organism whosedetection indicates a normal state or a particular disease state (forexample, the presence of a mRNA molecule may indicate an inflammation).The biomarker may therefore be used as an indicator of a particulardisease state or some other biological state of a tissue, an organ or anorganism. In the context of the present invention and not excluding theabove-mentioned applications, in situ hybridization may also be used,for example, to demonstrate and characterize the bioactivity of an agentor a combination of agents in intact organisms.

For in situ hybridization, the intact biological sample (tissues,organs, or whole organisms) or sections thereof are typically treated byorganic solvents to fix the target RNA transcripts in place and toincrease access of the probe. The probe is typically either a labeledcomplementary oligonucleotide, DNA or RNA (riboprobe). The probe, forexample, hybridizes to the target mRNA sequence at elevated temperature,and then the excess probe is washed away (after prior hydrolysis usingRNase in the case of unhybridized, excess RNA probe). As would beunderstood by those skilled in the art, solution parameters, such astemperature, salt and/or detergent concentration may be manipulated toremove any non-identical interactions (i.e. only exact sequence matcheswill remain bound). Then, the probe that was labeled with either radio-,fluorescent- or antigen-labeled bases (e.g. digoxigenin, or biotin) islocalized and quantitated in the tissue using either autoradiography,fluorescence microscopy, or immunohistochemistry, respectively. In situhybridization procedures may also involve the use of two or more probes,labeled with radioactivity or the other non-radioactive labels, tosimultaneously detect two or more transcripts in section of a tissue ororgan, or in the whole organisms (e.g. embryo, larvae, and tadpole). Thebasic steps of in situ hybridization procedures typically include, forexample, permeabilization of the biological specimen (tissue, organ,embryo) with proteinase K to open cell membranes, although thepermeabilization step is not always needed for tissue sections or someearly-stage embryos. Next, labeled complementary probes are allowed tobind to the target mRNAs by hybridization. Specific hybrids encompassingthe probe and the target mRNA are visualized by staining with anantibody, which recognizes a specific antigen (e.g. digoxigenin orbiotin) covalently coupled to the probe molecule. The antibody may beradioactively labeled, covalently coupled with a fluorophore orconjugated with an enzyme such as horse radish peroxidase. The enzyme isused to catalyze a color-producing reaction. Other catalytic enzymessuch as alkaline phosphatase may be used instead of peroxidases for bothdirect and indirect staining methods. Alternatively, the antibody may bedetected using the fluorescent label (by immunofluorescence), orradioactivity (by autoradiography). Fluorophores may include, withoutlimitation, fluorescein, rhodamine, DyLight Flour or Alexa Fluor. Otherdetection techniques, such as, for example, luminescence, may also beused in the methods of the invention.

In situ hybridization procedures of use in the instant methods may becarried out manually, semi-automated or fully automated. Manually refersto procedures carried out by solely hand without using specializedequipment (e.g. machines, instruments, or robots). Semi-automatedindicates that parts of procedures are done by specialized equipment,but human intervention may be required for one or more steps. Fullyautomated in situ hybridization procedures use specialized equipmentthat may be controlled fully or in part by computers.

For purposes of the methods of the instant invention,immunohistochemistry refers to the process of localizing antigens (e.g.proteins, lipids, glycolipids, or glycans) in cells of a tissue, organ,or intact organisms by exploiting the principle of antibodies bindingspecifically to antigens in biological samples. Immunohistochemicalsamples are, for example, whole organisms (e.g. embryos, tadpoles,larvae, adults), organs, or tissues or sections thereof.Immunohistochemical staining may be used in the diagnosis of normal aswell as abnormal cells such as those found in tissues and organs thathad undergone pathological changes due to cancer or other illnesses. Themolecular markers (e.g. antigens) detected by immunochemistry mayreflect the severity or presence of some disease state.Immunohistochemistry may also be used to understand the distribution andlocalization of biomarkers (e.g. differentially expressed antigens) indifferent parts of a biological sample. Specific biomarkers arecharacteristic of particular cellular events such as proliferation,differentiation, or cell death (apoptosis). A biomarker or molecularmarker can be an antigen native to the tissue, organ, or organism whosedetection indicates a normal state or a particular disease state (forexample, the presence of a protein may indicate an inflammation). Thebiomarker is therefore used as an indicator of a particular diseasestate or some other biological state of a tissue, an organ or anorganism. In the context of the present invention, and not excluding theabove-mentioned applications, immunohistochemistry may also be used todemonstrate the bioactivity of an agent or a combination of agents inintact organisms.

For purposes of the instant invention, the visualization of anantibody-antigen interaction may be accomplished in a number of ways, aswould be understood by those of skill in the art. For example, in themost common instance, an antibody is conjugated to an enzyme, such asperoxidase, that can catalyse a color-producing reaction(immunoperoxidase staining). Other catalytic enzymes such as alkalinephosphatase may be used instead of peroxidases for both direct andindirect staining methods. Alternatively, the antibody may be detectedusing a fluorescent label (immunofluorescence), may be attached tocolloidal gold particles for electron microscopy, or may be maderadioactive for autoradiography. Fluorophores of use in the instantmethods may include, without limitation, fluorescein, rhodamine, DyLightFlour or Alexa Fluor. Other detection techniques, such as, for example,luminescence, may also be used in the methods of the invention.

The antibodies used in the immunohistochemistry of the instant methodsmay be polyclonal, i.e. raised by normal antibody reactions in animals,such as horses, sheep or rabbits. Polyclonal antibodies (or antisera)are obtained from different B cell resources. They are typically acombination of immunoglobulin molecules secreted against a specificantigen, each identifying a different antigenic determinant (epitope).Alternatively, monoclonal antibodies can be used that have affinity forthe same antigen. Monoclonal antibodies are monospecific, because theyare made by one type of immune cell, which are all clones of a uniqueparent B cell.

Two basic strategies are typically used for the immunohistochemicaldetection of antigens in tissue, organs, or whole organisms: the directmethod and the indirect method. In both cases, many antigens may alsoneed an additional step for unmasking. This may be achieved by detergenttreatment or sectioning. The direct method is typically a one-stepstaining method, and normally involves a labeled antibody (e.g.fluorophore or enzyme conjugated antibody) reacting directly with theantigen in the biological sample. The indirect method typically involvesan unlabeled primary antibody (first layer), which reacts with theantigen, and a labeled secondary antibody (second layer), which reactswith the primary antibody. The second layer antibody may be conjugatedwith a fluorescent dye or an enzyme. The secondary antibody may also bebiotinylated and coupled with streptavidin-horseradish peroxidase orother streptavidine-enzyme fusion proteins. The enzymes are typicallyused to catalyze a color-producing reaction. Finally, the antibody mayalso be radiolabeled and antibody-antigen complexes are detected byradiography. Other detection techniques, such as, for example,luminescence, may also be used in the methods of the invention.

The immunohistochemical procedures of the instant methods may be carriedout manually, semi-automated or fully automated. Manually refers toprocedures carried out by solely hand without using specializedequipment (e.g. machines, instruments, or robots). Semi-automatedindicates that parts of procedures may be done by specialized equipment,but human intervention is required for one or more steps. Fullyautomated immunohistochemical procedures use specialized equipment thatmay be controlled fully or in part by computers.

In another aspect, the invention provides compounds identified by an invivo screening method comprising the steps of: a) treating a pluralityof amphibians with a plurality of agents; b) identifying an amphibianfrom the plurality of amphibians wherein the treatment causes edema inor death of the amphibian; c) determining the anatomical pattern ofedema formation in the identified amphibian; and d) identifying theagent causing the edema in or death of the amphibian. As described indetail in the Examples, methods of the instant invention have been usedto identify compounds causing various phenotypes in treated amphibians.

In some embodiments of the invention, the compound identified by the invivo screening method affects blood vessel development only. In morespecific embodiments, the compound causes defective vasculogenesis. Ineven more specific embodiments, the compound causes defectiveangiogenesis, ectopic angiogenic sprouting, or vitelline vein network(VVN) hypoplasia.

In some embodiments of the invention, the compound affects both bloodand lymph vessel formation. In specific embodiments, the compound causesdefective blood and lymph angiogenesis or vitelline vein networkhyperplasia and defective lymph angiogenesis.

In some embodiments of the invention, the compound affects lymph vesselformation only. In specific embodiments, the compound causes defectivelymph angiogenesis.

In another aspect, the invention provides methods for in vivo screeningcomprising: a) treating a plurality of amphibians with a plurality ofagents; b) identifying an amphibian from the plurality of amphibianswherein the treatment causes edema in or death of the amphibian; c)determining the anatomical pattern of edema formation in the identifiedamphibian; and d) identifying a pathway that mediates lymphatic and/orvascular development in the identified amphibian. In some embodiments ofthe invention, the pathway is a VEGF pathway. In some embodiments of theinvention, the pathway is targeted by an adenosine receptor antagonist.

In yet another aspect, the invention provides novel methods according tothe following numbered paragraphs:

1. A method for in vivo screening comprising: a) treating a plurality ofamphibians with a plurality of agents; b) identifying an amphibian fromthe plurality of amphibians wherein the treatment causes edema in ordeath of the amphibian; and c) determining the anatomical pattern ofedema formation in the identified amphibian.2. The method of paragraph 1, wherein the agent is administered to theamphibian by dissolving the agent in the culture media containing theamphibian.3. The method of paragraph 1, wherein the method is performed in amulti-well format.4. The method of paragraph 1, wherein the amphibian is an embryo,tadpole, or adult.5. The method of paragraph 1, wherein the amphibian is from the subclassLissamphibia.6. The method of paragraph 5, wherein the amphibian is a frog, toad,newt, salamander, mudpuppy, or caecilian.7. The method of paragraph 1, wherein the amphibian is from the genusXenopus.8. The method of paragraph 7, wherein the amphibian is from the speciesXenopus laevis or Xenopus tropicalis.9. The method of paragraph 1, wherein the agent is a small molecule, adrug, an antibody, a peptide, a secreted protein, a nucleic acid, anantisense RNA molecule, a ribozyme, an RNA interference nucleotidesequence, an antisense oligomer, or a morpholino oligonucleotide.10. The method of paragraph 1, wherein the edema or death is caused byan activity in the vascular, lymphatic, cardiac, or excretory system ofthe identified amphibian.11. The method of paragraph 1, further comprising the step ofidentifying the target tissue(s) or organ(s) of the agent responsiblefor the edema or death in the identified amphibian.12. The method of any one of paragraphs 1-11, wherein the identifyingstep is performed by a secondary screen.13. The method of any one of paragraphs 1-12, wherein the identifyingstep is performed by in situ hybridization.14. The method of paragraph 13, wherein the in situ hybridization isperformed manually, semi-automated or fully automated.15. The method of any one of paragraphs 1-12, wherein the identifyingstep is performed by immunohistochemistry.16. The method of paragraph 15, wherein the immunohistochemistry isperformed manually, semi-automated or fully automated.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES

Pathological neovascularization is associated with many severe anddebilitating diseases such as chronic inflammation, diabeticretinopathy, and cancer. Despite of significant advances inantiangiogenic therapies in recent years, studying the mechanisms ofblood and lymphatic vessel formation to define novel therapeutic targetsand the identification of novel anti(lymph)angiogenic drugs remainshighest priority. This is particularly true for lymphangiogenesis, wherethe mechanistic understanding and the identification of novel drugtargets have been hampered by a lack of an appropriate, simple animalmodel. The recent imaging and molecular characterization of lymphaticvessel systems in Xenopus tadpoles (Ny et al., 2005) and zebrafish(Kuchler et al., 2006; Yaniv et al., 2006) has created new opportunitiesfor studying vascular development as well as for pharmacologicalscreens. Here the inventors describe in viva chemical screening methodsto identify novel bioactive compounds and to define several novelpathways acting during blood vascular development and lymphangiogenesisin Xenopus tadpoles. In addition, the methods are suitable to identifycompounds modulating cardiac and renal development or functions in vivo.

Forward chemical genetics uses the screening of annotated libraries ofsmall organic compounds with experimentally-verified biologicalmechanisms and activities to study biological systems (Stockwell, 2000).This approach circumvents the well-known problems of targetidentification and lack of mechanistic understanding associated withactive compounds recovered from screens using conventional chemicallibraries (Root et al., 2003). Forward chemical genetics has thereforebecome increasingly used in cell cultures to identify signaling pathwaysinvolved in cellular functions in vitro (Diamandis et al., 2007:Rickardson et al., 2006; Root et al. 2003), and more recently, wholeorganisms such as Drosophila, Caenorhabditis elegans, and zebrafish havebeen used for compound discovery (Chang et al., 2008; Min et al., 2007;Tran et al., 2007). Importantly, chemical genetics using whole animalsoffers a complementary approach to loss-of-function mutations orknockdowns with siRNA or morpholino oligonucleotides in the analysis ofcomplex biological processes, such as organogenesis.

The use of Xenopus embryos for chemical library screening and drugdiscovery was previously proposed (Brandli, 2004), and subsequently asmall-scale pilot study provided further evidence for the feasibility(Tomlinson et al., 2005), but the phenotypic read-outs useful in theidentification of active agents were not understood, nor were phenotypicread-outs combined with secondary screens. The inventors now describe alarge-scale two-step chemical screening method in Xenopus embryos toefficiently recover agents affecting vascular, lymphatic, cardiac orrenal development and function in vivo (FIG. 1). As utilized herein, theagent or compound can be but is not limited to a chemical, a smallmolecule, a drug, an antibody, a peptide, a secreted protein, a nucleicacid (such as DNA, RNA, a polynucleotide, an oligonucleotide or a cDNA)or an antisense RNA molecule, a ribozyme, an RNA interference nucleotidesequence, an antisense oligomer or a morpholino oligonucleotides.High-throughput, large-scale screening approaches require a functionalphenotypic read-out that is easily detected and reliably predictive forvascular system defects. Edema is the major downstream phenotype causedby impairment of lymphatic vessel development (Ny et al., 2005), but isalso associated with disrupted development or function of thecardiovascular and excretory systems in tadpoles (Howland, 1916). Theinventors therefore decided to screen the LOPAC¹²⁸⁰ chemical library byvisual inspection for compounds that induce edema. There were severaldifferent types/locations of edema induced by different compounds (FIG.2). Remarkably, compounds targeting the same pathway often revealedsimilar phenotypes (Table 8). For example, Ca²⁺-channel antagonistsinduced pericardial and ventral edemas, whereas retinoids causedcerebral and pronephric edemas. Although most gene defects affectinglymphatic vessel development also cause edema formation, the possibilitythat some compounds might have affected lymphatic (or blood) vesseldevelopment without causing edema formation cannot be excluded. Theinventors therefore also scored for compounds causing late stagelethality in response to compound treatment. A total of 66 compoundssatisfying the screening criteria were recovered with a hit rate of 5%:48 edema-inducing compounds and 18 compounds causing lethality (Tables1, 3, and 4). From a practical point of view, the inventors were able toexclude 95% of the compounds as inactive on the basis of a simple,non-invasive phenotypic screening criteria.

Edema formation and lethality may not only be caused by cardiovasculardefects, but could also be a consequence of renal dysfunction impairingfluid homeostasis. In the second screening step, compound-treatedembryos were therefore subjected to semi-automated whole mount in situhybridizations using specific blood vascular and lymphatic marker genes.The analysis of vascular marker gene expression revealed that a total of32 hits (24 of the 48 edema-inducing compounds and 8 of the 18 compoundsassociated with lethality) caused abnormal vascular development andmorphogenesis in tadpoles. The 32 compounds represented 15 distinctpharmacological classes and they could be grouped into three broadvascular phenotype classes on the basis of affecting either bloodvascular or lymphatic development only or both (Table 9). Collectively,the phenotypic screening method resulted in recovery of bioactivecompounds with an impressive 49.2% hit rate (32 of 65 tested).Alternatively, the second screening step may be performed usingimmunohistochemistry in place of in situ hybridization.

The inventors also assessed whether known antiangiogenic compounds wererecovered in the Xenopus screen. The LOPAC¹²⁸⁰ library harbors eightcompounds (difluoromethylornithine, indirubin-3′-monoxime,2-methoxyestradiol, minocycline hydrochloride, SU 4312, SU 5416,thalidomide, tyrphostin AG1478) with known antiangiogenic activitivities(Serbedzija et al., 1999; Tran et al., 2007). Three antiangiogeniccompounds [difluoromethylornithine, minocycline hydrochloride,thalidomide] are not considered, since they are highly hydrophilic andtherefore poorly penetrate embryos (Tran et al., 2007). A screen of theLOPAC¹²⁸⁰ library using transgenic zebrafish expressing a fluorescentvascular reporter gene resulted in the recovery of three out of the fiveantiangiogenic compounds (indirubin-3′-monoxime, SU 4312, TyrphostinAG1478) (Tran et al., 2007). In Xenopus, all five compounds[indirubin-3′-monoxime, 2-methoxyestradiol, SU 4312, SU 5416, TyrphostinAG1478] scored as active in the phenotypic Xenopus screens (Tables 3 and4). Importantly, the two VEGFR inhibitors SU4312 and SU5416 wereidentified as positive hits validating the screening procedure (FIG. 5m, n). Taken together, the analysis demonstrates that the phenotypicchemical library screening method is capable of efficiently identifyingantiangiogenic compounds in Xenopus embryos. This occurs with higherefficiency and sensitivity than with the transgenic zebrafish reporterline. The phenotypic screening method is widely applicable to otheraquatic lower vertebrate animal models as it does not require thegeneration of transgenic reporter lines. With regard to amphibians, themethod is applicable to the subclass Lissamphibia, which includes allliving amphibians. Lissamphibia consist of three orders: Anura (frogsand toads), Urodela or Caudata (newts, salamanders, and mudpuppies), andGymnophiona or Apoda (caecilians). In a particular embodiment, theamphibian belongs to the genus Xenopus, which includes Xenopus laevis orXenopus tropicalis.

In the addition to the known antiangiogenic compounds, the inventorshave identified 27 new compounds affecting vascular and/or lymphaticdevelopment in Xenopus embryos. A number of these compounds, such asnocodazole, podophyllotoxin, forskolin, and retinoic acid, are known tohave broad effects on many cell types. Without intending to be bound bytheory, the antiangiogenic activities observed for these compounds arelikely the result of pleiotropic effects. The predicted biologicalfunctions of other recovered compounds implicate the requirement ofvarious mechanisms, including hormone and cyclic nucleotide signaling,phosphorylation as well as K⁺- and Ca²⁺-channels for the normaldevelopment of blood vessels and/or lymphatics. Regarding the hitsexhibiting only blood vascular system defects in vivo, this includedcompounds causing defective vasculogenesis, impaired angiogenesis, orVVN hypoplasia. Interestingly, the Ca²⁺ATPase inhibitor calmidazoliumchloride and Raf1 kinase inhibitor GW5074, two compounds not previouslyknown to exhibit activities on the vascular system, were found to act ina pro-angiogenic fashion by promoting ectopic, premature angiogenesis inXenopus embryos (FIG. 3 g,h, i, j). In addition, effects on the VVN werenoticed (FIG. 3 h, j). GW5074 was also tested in endothelial cellculture assays, where it promoted endothelial cell proliferation (FIG.6). On the basis of in vitro and in vivo evidence, but without intendingto be bound by theory, these compounds imply Ca²⁺-ATPase and Raf kinasein the regulation of endothelial cell function. Furthermore, and withoutintending to be bound by theory, these small-molecule compounds may havethe ability to substitute for mitogenic growth factors used to stimulateendothelial cell proliferation.

Several compounds inhibiting preferentially lymphatic vasculardevelopment were identified in the assays, including several tyrosinekinase inhibitors (SU 4312, SU 6656, GW2974), and L-type calcium channelblockers (felodipine, nicardipine) (Table 9, FIG. 5). Treatment with theVEGF- and PDGF-receptor antagonist SU 4312 had the most potent effect onlymphatic vessel formation in vivo as demonstrated by the absence ofVEGFR3-positive structures of the lymphatic system in Xenopus embryos(FIG. 5 m). The blood vasculature remained largely unaffected (FIG. 5n). These results indicate that, at the concentration of 20 μM used inthe Xenopus assay. SU 4312 targets in vivo primarily the VEGFC/VEGFR3signaling pathway, which regulates embryonic lymphangiogenesis intadpoles and mouse embryos (Karkkainen et al., 2004; Ny et al., 2005).At higher concentrations (>30 μM), SU 4312 will also exhibit moderateantiangiogenic effects as was recently demonstrated in zebrafish (Tranet al., 2007). Collectively, but without intending to be bound bytheory, these findings indicate that SU 4312 may act in vivo primarilyon lymphatic vessels. This also applies to the dual ErbB2 and EGFreceptor tyrosine kinase inhibitor GW2974 and Src family kinaseinhibitor SU 6656, which both exhibited antilymphatic activities invitro and/or in vivo.

The role of calcium channels in lymphatic vessel formation and functionhas not been studied to date. The calcium channel blockers felodidipineand nicardipine have vasodilatory effects and are therefore widely usedto treat hypertension. However, one bothersome side-effect of theseolder dihydropyridines is edema formation especially in the feet, legs,and ankles (Ram, 2006), which could indicate dysfunction of thelymphatic vasculature.

The majority of compounds with inhibitory effects on the vascular orlymphatic development in Xenopus also inhibited proliferation and/ortube formation of cultured human LEC and HUVEC (FIG. 6). 7-cyclo andnaphthyridine, two compounds disrupting both blood and lymph vesselformation in vivo, also exhibited inhibitory activities for both HUVECand LEC in vitro (FIG. 4 c-f; FIG. 7). Furthermore, the inhibition ofHUVEC tube formation in vitro correlates well with the in vivoobservation of defective intersomitic vein angiogenesis. Despite thesecompelling examples, many compounds with in vivo bioactivity showed noeffects in cell culture models indicating that the in vitro assays mayfail to adequately reproduce all steps of vascular development (Table10). The results indicate that in vitro cell-based chemical libraryscreens are less reliable and lack sufficient predictive power toidentify compounds with anti-angiogenic and/or -lymphatic activity invivo. In contrast, Xenopus tadpoles allow high-throughput screening in aphysiological context compared to traditional cell-based screens.

The inventors have also investigated the potential involvement ofcandidate compounds recovered from the Xenopus tadpole screens in(lymph)angiogenesis in mammals. Naphthyridine, an adenosine A1 receptorantagonists, inhibited blood and lymphatic vessel formation in tadpoles,which could also be recapitulated using in vitro endothelial cellproliferation and tube formation assays (FIG. 4 e, f, FIG. 6). In aproof-of-principle study, naphtyridine was tested in an establishedmouse model of VEGFA-induced dermal neovascularization (FIG. 8).Remarkably, systemic treatment of mice with naphthyridine potentlyinhibited lymphatic vessel enlargement and angiogenesis indicating thatadenosine A1 receptors do not only act antagonistically on amphibianvascular development but also disrupt adult mammalian(lymph)angiogenesis. The conservation of the in vivo activities betweenamphibians and mammals validates Xenopus embryos and tadpoles asrelevant screening tools in drug discovery for human diseases.

Vascular growth and function are regulated by a complex interplaybetween different cell types and mediators, which cannot be sufficientlyreproduced by in vitro culture systems. The use of Xenopus embryos hasseveral advantages over mouse models of vascular development: Tadpolesonly need little space and low-cost growth medium, and five tadpoles canbe incubated simultaneously per well of a 48-well plate. Since in theearly stages of development, nutrients, oxygen, and also small organicmolecules freely diffuse through the skin, injections are not necessary.Moreover, tadpoles are not highly motile; thus, no anesthesia is neededfor visual inspection. Importantly, treatment with small moleculesrevealed highly reproducible results, as the same phenotype was usuallyobserved in all embryos treated with the same compound. Visualinspection is rapid and a single investigator can easily analyze 320compounds per week. Zebrafish represents an alternative lower vertebrateanimal model for whole organism-based drug discovery screens as itshares many advantages with Xenopus (Zon and Peterson, 2005). Mostnotably, zebrafish mutants with vascular defects have been used inlarge-scale chemical suppressor screens to identify small moleculesconferring therapeutic benefits (Zon and Peterson, 2005). However, froman evolutionary perspective, amphibians are the animal models of choiceas they have co-evolved with mammals for almost 100 million years longerthan fish (Brändli, 2004). This fact may also provide an explanation forthe low hit rate observed in a recently reported zebrafish-based screenof the LOPAC library for compounds with anti-angiogenic activities (Tranet al., 2007) and may account for the failure to identify known andnovel antiangiogenic molecules, such as naphtyridine.

The instant examples demonstrate that the Xenopus embryo model incombination with a two-step screening method represents an effective,low-cost platform to identify novel pathways involved in angiogenesisand lymphangiogenesis, and to accelerate the discovery of noveldrug-like small organic molecules. Despite the obvious differences inconstitution and physiology between amphibians and humans, the Xenopustadpole model represents a much-needed tool to bridge the gap in drugdiscovery between traditional in vitro and preclinical animal models.

Materials and Methods a) Xenopus Embryo Husbandry

In vitro fertilization and culture of wild-type and albino Xenopusembryos were performed as previously described (Brands and Kirschner,1995; Helbling et al., 1998). Embryos were raised in 0.1×MMR (1×MMR: 0.1M NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM Hepes, pH 7.8) at roomtemperature and staged according to Nieuwkoop & Faber (Nieuwkoop andFaber, 1994). Any unfertilized, dead or malformed embryos were removedfrom the embryo cultures.

b) Chemical Library Screening and Confirmatory Testing

The LOPAC¹²⁸⁰ chemical library (Sigma, #LO2800, Lot No. 035K4701)containing 1,280 compounds (10 mM in DMSO) was used for the phenotypescreening of Xenopus embryos. 10-μl aliquots of the chemicals weretransferred from the mother plates with a liquid handling robot(Aquarius, Tecan) into 96-well tissue culture plates to generate dilutedstock plates (2 mM in DMSO; 50 μl final volume). Wild-type, healthyXenopus embryos were arrayed at stage 29-30 (35 hours postfertilization, hpf) into polystyrene flat-bottom 48-well tissue cultureplates (BD Falcon #353078) (5 embryos per well) containing 1 ml 0.1×MMR.Once embryos reached stage 31 (37 hpf), 10-μl aliquots of the dilutedchemicals were added to the wells. The final concentration of eachchemical was 20 μM in the presence of 1% DMSO. The γ-secretase inhibitorcompound E (Merck #565790; 20 μM) was used as a positive, edema-inducingcontrol compound (R.E.K. & A.W.B, unpublished observation). Negativecontrol wells contained embryos in screening medium and 0.1×MMR only.Embryos were treated with the chemicals in a humidified incubator at 23°C. over a timeframe of 4 days. Embryos were manually scored for thepresence of edema or other morphological phenotypes using a teachingdissecting microscope (SV6; Carl Zeiss). Two investigators performedseparately the scoring, daily at stages 39 (56 hpf), 41 (76 hpf), 45 (98hpf), and 47 (120 hpf). Dead embryos were removed and 100 μl of waterwas added daily to each well to compensate for fluid evaporation.Chemicals were considered to be active when at least 4 out of 5 embryosdisplayed the same phenotype (edema, lethality, or other phenotypes).All putative hits were retested and in each case comparable results wereobtained. For imaging at the end of the experiments, Xenopus tadpoleswere anesthetized with 0.05% tricaine (Sigma #A5040) in 0.1×MMR. Imageswere captured using a stereomicroscope (SteREO Lumar V12; Carl Zeiss)equipped with a digital camera (AxioCam Color; Carl Zeiss).

c) Secondary Chemical Screening

In follow-up studies, 65 chemicals from the LOPAC¹²⁸⁰ library thateither induced edema formation or lethality were tested for theirability to interfere with blood vessel development and/orlymphangiogenesis in Xenopus embryos. All chemicals were purchased fromSigma and 10-mM stock solutions in DMSO were prepared. Chemicals wereused at a final concentration of 20 μM in screening medium to treathomozygous albino Xenopus embryos, which lack natural pigmentation.Chemical treatments of embryos (n=7 per well) was initiated at stage 31(37 hpf) and terminated at stages 35/36 (50 hpf) or 42 (80 hpf). Embryoswere fixed in 4% paraformaldehyde and processed for whole mount in situhybridization.

d) Whole Mount In Situ Hybridizations

Whole mount in situ hybridizations and synthesis of dixoxigenin-labeledin situ hybridization probes were performed as described previously(Helbling et al. 1999; Saulnier et al., 2002). Where albino embryos wereused, the bleaching step was omitted. Semi-automated whole mount in situhybridizations were performed using the Biolane HTI machine (Hölle &Hüttner AG, Germany). Digoxigenin-labeled cRNA probes were generatedfrom linearized plasmids encoding the blood vessel marker apj (Kälin etal., 2007) and lymph vessel marker vegfr3 (GenBank Acc. No. BM2611245).Sense strand controls were prepared from the plasmids and then testednegative by in situ hybridization. Images were captured using astereomicroscope (SteREO Lumar V12; Carl Zeiss) equipped with a digitalcamera (AxioCam Color; Carl Zeiss). Composite figures were organized andlabeled using Adobe Photoshop CS2 and Adobe Illustrator CS software.

e) Mammalian Cell Culture

Human umbilical vein endothelial cells (HUVEC) were purchased fromPromoCell. Dermal lymphatic endothelial cells (LEC) were isolated fromneonatal human foreskins and characterized as described previously(Hirakawa et al., 2003; Kajiya et al., 2005). HUVEC and LEC werecultured in endothelial basal medium EBM (Cambrex) supplemented with 20%fetal bovine serum (Invitrogen), antibiotic-antimycotics (100 U/mlpenicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin; Invitrogen),2 mM L-glutamine (Invitrogen), 10 μg/ml hydrocortisone (Fluka) and 25μg/ml N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt(Fluka) for up to eleven passages. LEC cultures were validated forlineage-specific cell differentiation by quantitative real-time RT-PCR(lymphatic vascular marker genes: PROX1, LYVE1, and PODOPLANIN1; bloodvascular endothelial marker genes: VEGFR1 and VEGFC) and byimmunocytochemistry (marker tested: CD31, LYVE1, and PROX1) as describedpreviously (Hirakawa et al., 2003).

f) Chemical Treatments of Cell Cultures

Proliferation and tube formation assays were largely performed asdescribed (Kajiya et al., 2005). To test for effects on cellproliferation, HUVEC and LEC (3×10³ cells) were seeded into fibronectin(10 ug/ml)-coated flat-bottom black 96-well tissue culture plates(#3603, Corning) and treated with selected chemicals at a screening doseof 10 μM in 0.1% DMSO or with 0.1% DMSO only. After 48 hours at 37° C.,cells were incubated with 4-methylumbelliferylheptanoate (Sigma #M2514)as described (Kajiya et al., 2005). The intensity of fluorescence, whichis proportional to the number of viable cells, was measured using amicroplate reader (SpectraMax Gemini EM; Molecular Devices). Thechemical treatments were performed in five replicates.

The effect of the selected chemicals on the formation of tube-likestructures was performed as follows. Confluent monolayers of HUVEC andLEC were overlaid with 0.5 ml of neutralized isotonic bovine dermalcollagen type I (1 mg/ml for LEC, 1.2 mg/ml for HUVEC; PureCol #5409,Inamed BioMaterials) containing either the selected chemical (1 μM in0.1% DMSO) or vehicle only (0.1% DMSO). For7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine (naphthyridine), the IC50value on LEC tube formation was determined using the following compounddoses: 0.2 μM, 0.4 μM, 0.55 μM, 0.7 μM, 1 μM, and 2 μM. After overnightincubation at 37° C., the treated cell cultures were fixed with 4%paraformaldehyde. Each chemical treatment was performed in triplicateand the experiment was repeated at least three times. Threerepresentative images of hotspots with tube-like structures were takenper well using a digital camera (AxioCam MRm, Carl Zeiss) mounted on aninverted microscope (Axiovert 200M, Carl Zeiss). The length of thetube-like structures was measured using the IPLab software (BDBiosciences). For each experimental condition, results are representedas average total tube length (μm) per area (three image fields).

g) Matrigel Plug Implantation Assay

The mouse studies were conducted under protocols approved by theVeterinary Office of the Canton of Zurich, Switzerland (permit#123/2005). Nine week-old female wild-type FVB mice (Charles River) wereused for the experiments (n=6 per group). Prior to Matrigelimplantation, the mice were anesthetized by intraperitoneal injection of200 μl of Dormitor (20 μg/ml medetomidine; Pfizer) and Narketan 10 (8mg/ml ketamine; Vétoquinol). The left hip of the animals were shaved andsubsequently Matrigel was implanted by intradermal injection of 100 μlof unpolymerized growth factor-reduced Matrigel (BD Biosciences)containing 0.5 mg/ml recombinant human VEGFA (#0081109; National CancerInstitute, USA). The mice were treated orally with either 200 μl PEG-400(Fluka; pH adjusted to 5.2; control group) or with 200 μl PEG-400containing 71.5 μg naphthyridine (3 mg/kg/day). Another control group ofmice received Matrigel without VEGFA and was treated with 200 μlPEG-400. The treatment was given once daily for six days. Weight andappearance of animals was monitored daily. On the 7^(th) day, theMatrigel implants were removed from the euthanized mice forimmunocytochemical examination. The Matrigel implants were frozen inTissue-Tek O.C.T. compound (Sakura Finetek), sectioned, and processed asdescribed previously (Kajiya et al., 2005). In brief, 6-μm frozensections were fixed in −20° C. acetone for two minutes and in cold 80%methanol for five minutes, followed by incubation with antibodiesagainst the lymphatic-specific marker LYVE1 (1:1,000; Millipore) andagainst the pan-endothelial marker CD31 (anti-mouse: 1:50; BDBiosciences). Corresponding secondary antibodies were labeled with Alexa488 or Alexa 594 (Invitrogen). Nuclei were counterstained with 20 μg/mlHoechst 33342 (Invitrogen). Sections were examined under an Axioskop 2mot plus microscope and digital images were taken using an AxioCam MRccamera (Carl Zeiss). Three pictures each were taken of vascularizedareas around the Matrigel implants (maximal distance from the implant:500 μm). The vessel density, average vessel size and the average tissuearea occupied by vessels were determined in CD31/LYVE1 stained sectionsusing the IPLab software (BD Biosciences) as described (Kajiya et al.,2005).

h) Statistical Analyses

Results from in vitro cell assays are represented as mean±standarddeviation (SD) of independently performed assays. Statistical analysiswas performed by comparing means of biological replicates using theunpaired two-tailed student's t-test (Excel. Microsoft). Resultsobtained from the in vivo mouse experiments are shown as means±standarderror of mean (SEM). The statistical significance was determined usingthe unpaired two-tailed student's t-test (GraphPad Prism Version 4;GraphPad Software). A value of P<0.05 was considered as significant.

Example 1 A Chemical Library Screen Identifies a Subset of Compoundswith Pharmacological Activity in Xenopus Embryos

A two-step whole organism-based chemical screening method was developedto rapidly identify novel small-molecule modulators of angiogenesis andlymphangiogenesis during Xenopus embryogenesis (FIG. 1). The Library ofPharmacologically Active Compounds (LOPAC¹²⁸⁰, Sigma-Aldrich) comprising1,280 bioactive compounds was selected for the embryo-based screenings.The annotated compound library consists of marketed drugs, faileddevelopment candidates, and gold standards that have well-characterizedactivities. The compounds represent 56 pharmacological classes withdiverse and experimentally validated biological mechanisms, such asG-protein coupled receptors (GPCR) and kinases (Rickardson et al.,2006).

The primary screening method used edema formation in compound-treatedtadpoles as a rapid phenotypic read-out. Edemas present as abnormalfluid-filled swellings in any organ of the body. They are caused byimbalanced fluid homeostasis either by increased secretion of fluid intothe interstitium or impaired removal of this fluid. The underlyingpathophysiological causes include increased hydrostatic pressure orreduced oncotic pressure in the circulatory system, obstruction of thelymphatic system, and retention of sodium and water. Edema formation cantherefore be used as a convenient indicator of either impairedcardiovascular, lymphatic, and/or excretory system functions.

Xenopus late tailbud embryos at stage 31 (37 hpf) were selected forcompound treatment. This embryonic stage coincides with the onset oflymphatic system formation and intersomitic vein angiogenesis duringXenopus embryogenesis (Helbling et al., 2000; Ny et al. 2005). Embryoswere arrayed into 48-well dishes (five per well) and treated with testcompounds at a concentration of 20 μM in the presence of 1% DMSO. As apositive control, embryos were treated with compound E, a cell-permeableinhibitor of γ-secretase (Seiffert et al., 2000), which potently inducesedemas (data not shown). All 48-well dishes also contained replicates ofnegative controls (1% DMSO). Embryos were monitored daily over a periodof four days for edema formation. In addition, drug-induced lethalityand other externally visible phenotypes were scored. On average, 320compounds were screened per week. The phenotypes observed were highlyreproducible as all embryos treated with a specific compound showed thesame phenotype. Furthermore, all of the compounds generating hits wereretested and yielded comparable results.

The majority (1,166 compounds; 91%) of the 1,280 compounds tested didnot cause any discernable phenotypes in embryos and tadpoles until stage47 (120 hpf), when treatments were terminated (Table 1). The remaining114 (9%) compounds scored as hits. Fifty compounds representing 4% ofthe LOPAC¹²⁸⁰ library caused either embryonic or lethality withoutevidence of edemas. The first group included 32 compounds that wereseverely cytotoxic resulting in early embryonic lethality within 14hours of compound treatment (Table 2). The second group consisted of 18compounds that were lethal in tailbud embryos and/or tadpoles from atstage 37/38 (54 hpf) onwards (Table 3). Finally, 64 (5%) compoundscausing specific, externally discernable phenotypes in embryos wereidentified. Edemas were observed after treatment with 48 (4%) of the1.280 compounds tested (Table 4). In addition, 10 compounds affectedskin pigmentation (Table 5) and 6 compounds caused other phenotypicchanges (Table 6).

Example 2 Compounds with Comparable In Vivo Activities Target DistinctPharmacological Pathways

The 1,280 compounds in the LOPAC library represent 56 distinctpharmacological classes of which 34 (61%) were active in the Xenopusscreen (Table 7). Among the 114 active compounds, those affectingphosphorylation (30: 26%) were most prominently represented, followed by9 (8%) interfering with the dopamine pathway, and 8 (7%) modulating Ca⁺channels. For seven pharmacological classes, which includephosphorylation, dopamine, and hormone signaling, a given compound wasable to contribute to one of three distinct phenotype classes. 13compound classes were contributing to at least two phenotype classes,whereas 14 were associated with a single phenotype class only. Thelatter included, for example, several representatives of the adenosineand cyclic nucleotide classes, which specifically caused edema intadpoles.

Characteristic sets of active compound classes were associated with eachphenotype class. For the sake of simplicity, only compound classes withtwo or more hits will be mentioned here in detail. Compounds causingcytotoxicity included modulators of phosphorylation (11 hits),intracellular calcium (2), leukotriene (2), and lipid signaling (2)(Table 2, Table 7). These cytotoxic hits represent 12-25% of the totalcompounds of their respective pharmacological classes. Compoundsinducing lethality were mainly derived from the phosphorylation (6hits), Ca²⁺ channel (2), and neurotransmission (2) classes with hitrates ranging from 4-11% (Table 3, Table 7). The 48 edema-inducingcompounds represent the largest subgroup of the 114 active compoundsidentified in the screen. Of these, modulators of phosphorylation (13hits), Ca²⁺ channels (6), cyclic nucleotides synthesis (3),transcription (3), as well as adenosine (3) and dopamine (3) signalingwere the most prominent (Table 4, Table 7). This suggests thatdysregulation of numerous signaling pathways contributes to edemaformation. Finally, 50% of the compounds causing pigmentation defects intadpoles were modulators of dopamine receptor signaling (5 hits; Table5. Table 7). This finding is remarkable as it is consistent with thewell-known role of dopaminergic neurons in the regulation ofpigmentation in Xenopus tadpoles (Dulcis and Spitzer, 2008).Furthermore, it indicates that the experimental parameters chosen forthe embryo-based chemical screen were optimal and sufficiently sensitiveto recover agents targeting in vivo pharmacological pathways of knownbiological significance.

Example 3 Shared Anatomical Patterns of Edema Formation Arise fromPharmacological Interference of Distinct Sets of Molecular Pathways

Edema formation in response to compound treatment may initially occurhighly regionalized, i.e. restricted to a specific organ or tissue,before becoming generalized to the whole body. Hence, the anatomicaldistribution and the temporal onset of edemas induced by the 48edema-inducing compounds were analyzed in greater detail. In particular,the question of whether compounds producing similar phenotypes interferewith one common molecular pathway or affect several ones was examined.Overall, it was observed that the initial development of edemas wasrestricted to distinct locations of the embryo. The most frequentlocations were the pericardial and ventral areas, followed by theperiocular and pronephric areas. For most compounds (39 out of 48), morethan one tissue was affected by edema formation. This indicates thatmultiple target tissues exist for a given compound. On the basis ofshared location of edema formation and severity of the observedphenotype, six phenotype classes were defined (Table 8). Importantly,each phenotype class was associated with a specific subset ofedema-inducing compounds that in turn define unique sets of essentialmolecular pathways.

One third of the edema-inducing agents (16 compounds) gave rise to thephenotype class A (Table 8). These compounds induced both pericardialand ventral edemas (FIG. 2 a-f). Frequently, a third site of edemaformation, typically in periocular or pronephric locations, was alsonoticed (FIG. 2 b, d). Periocular, pericardial, ventral, and pronephricedema often fused over time resulting in the formation of large,liquid-filled edemas occupying the entire abdomen of affected tadpoles(not shown). In addition, tadpoles treated with GW2974 or MRS 1845manifested also with enlarged lymph hearts (FIG. 2 d, f). Finally, theadenylate cyclase activator forskolin caused the class A-characteristicpericardial, ventral, and proctodeal edemas, which were howeveraccompanied by a dramatic shortening of the anterior-posterior body axis(FIG. 2 r). In total, the class A edema-inducing compounds represented10 distinct pharmacological mechanisms (Table 8). Most prominently, theyincluded all six edema-inducing Ca²⁺ channel blockers and two out of thethree adenosine receptor A1 antagonists present in the chemical library.

Embryos of class B manifested with periocular and ventral edemas, and,frequently, also developed pronephric edemas (Table 8: FIG. 2 g, h).Typically, compound treatment did not result in lethality. The 12compounds inducing this particular edema phenotype targeted 7 distinctmolecular pathways. Modulators of phosphorylation (5 hits) andantagonists of dopamine signaling (2 hits) were the most prominentlyrepresented pharmacological classes.

Pericardial edemas alone were the hallmark of phenotype class C (FIG. 2i, j). Edema formation occurred as early as stage 33/34 followed bylethality usually before stage 45. The compounds affected eight distinctmolecular pathways and include inhibitors of phosphorylation (2 hits)and compounds targeting the cytoskeleton (2 hits; Table 8). Phenotypeclass D was defined by highly characteristic periocular edemas that wereaccompanied by other sites of edema formation. Increased fluidaccumulation in periocular edemas caused subsequently the protrusion orbulging out of the eyes, a condition reminiscent of exophthalmos (FIG. 2k, l). The active compounds targeted three distinct mechanisms:apoptosis, gene regulation, and phosphorylation.

Embryos falling into phenotype class E were characterized by thepresence of both cerebral and pronephric edemas (Table 8; FIG. 2 m, n).Interestingly, all three compounds target retinoic acid receptors (RAR)suggesting that this edema phenotype is caused by dysregulation of RARsignaling. Finally, phenotype class F represents edema-inducingcompounds that manifested with a heterogeneous range of edema types(Table 8; FIG. 2 o-q). For example, genistein induced edemas localizedto the tail tip (FIG. 2 q). Despite the heterogeneity of edemas, allcompounds were found to interfere with a single pharmacological class,protein phosphorylation.

Taken together, the analysis indicates that a given edema phenotype mayarise from dysregulation of one or more distinct molecular pathways asdemonstrated by the use of defined pharmacological agents. In addition,the fact that compounds targeting the same common molecular pathway(i.e. Ca²⁺ channels and adenosine A1 receptors) frequently manifest withthe same characteristic edema phenotype further underscores the validityand robustness of the whole-embryo based chemical library screeningmethod.

Example 4 Small-Molecule Compounds Affecting Blood Vessel Developmentand Angiogenesis

48 compounds that induced edema in Xenopus tadpoles were identified(Table 2. Table 8) and 18 causing lethality at stage 37/38 (54 hpf) orlater (Table 3). Together, the 66 compounds comprise 5.1% of allcompounds present in the LOPAC library. Edema formation or late-stagelethality may result from excretory system defects, (cardio)vascularand/or lymphatic vascular pathologies. Semi-automated whole-mount insitu hybridization was next used to visualize the development of bloodand lymphatic vessels in Xenopus embryos treated with hits from theprimary screen. apj and vegfr3 (flt4) were used as marker genes for theblood (Kalin et al., 2007) and lymphatic vasculature (Ny et al., 2005),respectively. Xenopus embryos were treated at stage 31 with 65 selectedcompounds at a concentration of 20 μM each. Embryos were fixed for insitu hybridization either at stage 35/36 to analyze vasculogenesis andintersomitic vein angiogenesis defects or at stage 42 to evaluatepossible defects in lymph vessel development (FIGS. 3-5). It was foundthat 32 (49%) out of 65 compounds tested interfered with lymphaticand/or blood vascular development. 18 compounds blocked selectivelyblood vessel development, six compounds interfered with both blood andlymphatic vessel development, and eight compounds affected lymphaticdevelopment only (see Table 9 for summary).

With regard to the blood vascular defects, we identified four differentclasses: (1) Inhibition of vasculogenesis, as evidenced by hypoplasia ofthe vitelline vein network (VVN) and posterior cardinal veins (PCV) andabsence of intersomitic veins (ISV); (2) Inhibition of angiogenesis, asreflected by defective ISV outgrowth and normal PCV; (3) Ectopicangiogenic sprouting of the ISV and VVN defects; and (4) hypoplasia ofthe VVN.

Compounds that interfered with blood vessel vasculogenesis and PCVassembly included the casein kinase 1 inhibitor IC 261 (FIG. 3 a, b) andnocodazole, which disrupts cytoskeleton assembly (FIG. 3 c, d). Both ofthese compounds also impaired development of the ISV and VVN (equivalentto the extraembryonic vasculature of the avian chorio-allantoic membraneand the mammalian yolk sac). Similar effects were observed aftertreatment with the tyrosine kinase inhibitor tyrphostin AG 494,2-methoxyestradiol, the cytoskeleton inhibitor podophyllotoxin, theCdc25 phosphatase inhibitor NSC 95397, and the dual cyclooxygenase and5-lipoxygenase inhibitor meclofenamic acid (Table 9).

Angiogenesis inhibitors, as evaluated by the ability of compounds toblock ISV outgrowth, included indirubin-3′-oxime, a cyclin dependentkinase inhibitor (FIG. 3 e, f), and the VEGF receptor phosphotyrosinekinase inhibitor SU 5416 (Table 9), which is in agreement with previousstudies in zebrafish (Bayliss et al., 2006; Pamg et al., 2002; Tran etal., 2007). Retinoic acid, the adenylate cyclase activator forskolin,and the angiotensin receptor I agonist L-162,313 were further identifiedas potent inhibitors of developmental angiogenesis (Table 9).

Two compounds induced VVN defects and promoted premature angiogenicsprouting of intersomitic veins. After treatment with the Raf1 kinaseinhibitor GW5074 (FIG. 3 g, h), the VVN was dysplastic and—instead offorming a regular vessel network—the blood vascular endothelial cellswere dispersed throughout the ventral parts of the embryo. Moreover,thin ectopic ISV sprouting was evident in the posterior parts of the PCVwhen compared to DMSO-treated control embryos (FIG. 3 g, h, o, p). Thisectopic sprouting phenotype was even more pronounced after treatmentwith the calmodulin-dependent Ca²⁺ ATPase inhibitor calmidazoliumchloride (FIG. 3 i, j, o, p). In addition, the blood vesseLs, includingthe VVN, were larger and fused, representing hyperplastic vasculature.This hyperplastic endothelial phenotype is reminiscent of the phenotypeobserved after overexpression of vegfa in Xenopus embryos (KOn et al.,2007).

A fourth group of compounds only interfered with the assembly of theVVN. The leukotriene synthesis inhibitor MK-886 led to hypoplasia of theVVN, but did not interfere with PCV assembly and ISV outgrowth (FIG. 3k, l). Similarly, treatments with the NO-independent guanylyl cyclaseactivator YC-1 (FIG. 3 m, n), resulted in dispersed, punctuated patternsof VVN endothelia, which might indicate a block of endothelia cellproliferation or VVN assembly (Pyriochou et al., 2006). Other compoundsof this group include the phosphodiesterase III/IV inhibitorzardaverine, and the cyclin-dependent protein kinase inhibitorpurvalanol A (Table 9).

Example 5 Small-Molecule Inhibitors of Blood and Lymphatic VesselDevelopment

Both blood and lymphatic vessel malformations were noticed in Xenopusembryos and tadpoles after treatment with seven compounds (Table 9). Innormal control embryos, three primary sites of lymphatic systemdevelopment can be observed: the anterior lymph sacs (ALS) of the head,the region of the anterior lymph hearts (ALH) in the trunk, and theposterior lymph vessels (PLV) in the tail (FIG. 4 b). Along with theanalysis for blood vessel defects, these areas in compound-treatedembryos were assessed specifically for abnormalities in early lymphaticvessel development. Based on this differential analysis, two generalcompound-induced phenotype classes were identified: compounds affectingboth blood vessel and lymph vessel formation, and compounds disruptingselectively lymph vessel development only (Table 9).

The former phenotype class was comprised of six compounds. The srcfamily tyrosine kinase inhibitor7-cyclopentyl-5-(-4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamin(in short: 7-Cyclo) interfered with angiogenic ISV sprouting but notwith VVN assembly (FIG. 4 c). Formation of ALS and ALH rudimentsoccurred, but lymphangiogenesis was strongly suppressed and the lymphvessel assembly in the tadpole tails was disrupted (FIG. 4 d). The A1adenosine receptor antagonist7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine (in short: naphthyridine)interfered not only with ISV angiogenesis but also with VVN assembly(FIG. 4 e). At later stages, tadpoles displayed stunted, disorganizedlymphatic vessels arising from the ALS and ALH, and only a few LECs, butno lymph vessels, were detected in the tail (FIG. 4 f). Similar vascularphenotypes were observed after treatment with the JNK inhibitor SP600125(Table 9). A subgroup of three compounds manifested withlymphangiogenesis defects accompanied by VVN hyperplasia. Treatment withthe adenosine A1 receptor antagonist 1,3-Diethyl-8-phenylxanthine or thepoly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide causedhyperplasia of the VVN, while ISV angiogenesis occurred unaffected (FIG.4 g, not shown). Interestingly, lymphangiogenesis was largely suppressed(FIG. 4 h, not shown). Treatment with the tyrosine kinase inhibitorgenistein induced a severe blood vessel phenotype with stunted ISVs andhyperplastic VVN (FIG. 4 i). At later stages, blood vessels were readilydetected, whereas lymphatic vessels and LECs were absent (FIG. 4 j).This suggests that genistein may interfere with the specification oflymphatic cell lineages.

Example 6 Small-Molecule Inhibitors of Lymphatic Vessel Development

Eight compounds that specifically inhibited lymphatic vessel developmentwithout affecting the blood vasculature were recovered from the chemicallibrary screen (Table 9; FIG. 5). Each compound induced highlycharacteristic lymphatic defects ranging from subtle regional lymphvessel dysplasia (FIG. 5 c) to severe, widespread disruption of lymphvessel development (FIG. 5 i, k, m). The L-type calcium channel blockerfelodipine caused abnormal, dysplastic lymphatic vessel sproutingwithout affecting other areas of lymphangiogenesis (FIG. 5 c). Treatmentwith the DNA topoisomerase II inhibitor sobuzoxane and the Ca²⁺ channelblocker nicardipine resulted in the formation of discontinuous,dysplastic lymphatic vessels (FIG. 5 e, not shown). In addition, vegfr3expression persisted in the VVN of subuzoxane-treated embryos (FIG. 5e). The src family kinase inhibitor SU 6656 and the K⁺ channel blockerdequalinium dichloride resulted in embryos with poorly developed,stunted lymphatics emerging from the ALS and ALH and hypoplastic PLV(FIG. 5 g, not shown). The most severe defects in lymphatic vesseldevelopment were observed with three compounds—the inhibitor of proteinprenylation mevastatin, and the tyrosine kinase inhibitors GW2974 and SU4312 (FIG. 5 i-m). After compound treatment, the three primary sites oflymphatic vessel development (ALS, ALH, and PLV) were detectable, butlymphangiogenesis was largely suppressed and the assembly of lymphaticsin the tail was disrupted. This phenotype was most pronounced aftertreatment with SU 4312, which is known as a VEGF receptor-1/-2 (VEGFR)inhibitor (FIG. 5 m). Interestingly, blood vascular development iscritically dependent on VEGFR-1/-2 signaling, but appears to beunaffected in SU 4312-treated embryos as demonstrated by normalangiogenic ISV outgrowth (FIG. 5 n). This indicates that, at theconcentrations used here, SU 4312 selectively disrupts lymphangiogenesisby inhibiting VEGFC/VEGFR-3 signaling in vivo.

Example 7 Effects of Small-Molecular Compounds on In Vitro EndothelialCell Proliferation and Tube Formation

The whole-organism based chemical library screens resulted in theidentification of 32 compounds that interfered with lymphatic and/orblood vascular development in Xenopus tadpoles. It was next askedwhether the in vivo activities of the compounds extended also tomammalian endothelia and whether the compounds interfered directly withendothelial cell functions. To address these points, 24 compounds wereselected, and proliferation and tube formation assays, two importantsteps in vessel formation, were conducted using human lymphaticendothelial cell (LEC) and umbilical vein endothelial cell (HUVEC)cultures. Compounds were selected for in vitro testing on the basis thatthey represented different classes of in vivo active compounds (Table10). The list included 19 compounds inducing edema in Xenopus tadpolesand 4 compounds (indirubin-3′-oxime, MK-886, SP600125, and SU 5416)causing lethality. Finally, the sigma 1 receptor ligand L-687,384 waschosen as it did not affect vascular development in Xenopus embryos.

The cell proliferation assays were performed by treating the endothelialcell cultures with compounds at a screening dose of 10 μM in 0.1% DMSOfor 48 hours (FIG. 6 a; Table 10). Nine compounds were scored as havingmarginal or no effects in the endothelial cell proliferation assays(85-115% of control) regardless of the cell type tested. Nine compoundsmoderately promoted cell proliferation (>115-150% of control). Thisincludes three compounds (GW2974, retinoic acid, sobuzoxane) stimulatingendothelial cell proliferation in both cell types tested. Five compounds(L-687,384, MK-886, MRS 1845, naphthalimide, SU 4312) promotedproliferation in HUVEC but not LEC cultures; and only one, GW5074, wasselective for TEC cultures. Six compounds decreased endothelial cellproliferation in vitro. Podophyllotoxin and nocodazole inhibitedmoderately (65-82% of control), whereas naphthyridine stronglysuppressed (42-48% of control) LEC and HUVEC proliferation.Interestingly, the remaining three compounds disrupted proliferation ina cell type-specific manner. IC 261 and 2-methoxyestradiol inhibitedpreferentially LEC proliferation, whereas indirubin-3′-oxime selectivelyblocked HUVEC proliferation consistent with a previous report (Tran etal., 2007).

The effects of the selected compounds on endothelial tube formation wasassessed after overnight treatment of LEC and HUVEC cultures at acompound concentration of 1 μM in 0.1% DMSO. The length of the tube-likestructures was measured using the IPLab software. 14 out of 24 compoundstested had comparable effects in both cell types tested. These includefour compounds (cyclosporin A, nicardipine, sobuzoxane, SU 4312) witheither no or only marginal effects on tube formation (85-115% of controltube length) (FIG. 6 b). As an example, nicardipine is shown in FIG. 7.Moderate inhibition of tube formation (50-85% of control) in both celltypes was observed with seven compounds (genistein, GW2974, IC 261.L-687,384, naphthyridine, MK-886, retinoic acid) (FIG. 6 b). Finally,three compounds (7-cyclo, nocodazole, podophyllotoxin) were identifiedas strong inhibitors of endothelial tube formation (<35% of control)irrespective of the cell type tested (FIG. 6 b, 7). The remaining tencompounds showed differential, cell-type specific effects on tubeformation. Four compounds acted preferentially on HUVEC cultures.Phenylxanthine promoted HUVEC tube formation (123% of control for HUVECversus 90% for LEC) (FIG. 7). In contrast, indirubin-3′-oxime,felodipine, and 2-methoxyestradiol blocked tube formation preferentiallyin HUVEC cultures. This was most apparent after 2-methoxyestradioltreatments when HUVEC tube formation was 40% of control, while LECcultures were only moderately affected (80% of control) (FIG. 7).Interestingly, none of the compounds tested selectively promoted LECtube formation, but six compounds (GW5074, MRS 1845, naphthalimide,oligomycin A, SP600125, SU 5416, tyrphostin AG 494) preferentiallyblocked tube formation in LEC rather than HUVEC cultures (FIG. 6 b). Thedifferences were most striking with SP600125 (59% of control for LECversus 110% for HUVEC).

A comparison of the results from the in vitro and in vivo compoundscreens is shown in Table 10. Interestingly, only two compounds(cyclosporin A, nicardipine) at the concentrations tested here hadlittle or no activity in the in vitro assays, indicating that many ofthe in vivo active compounds also interfered with endothelial cellfunctions in vitro. The predictive power of the in vitro assays washowever limited. For example, three out of the five compounds affectinglymphangiogenesis in vivo had no activity in LEC-based in vitro assays.Notably, this included also the VEGF receptor antagonist SU 4312, whichrobustly blocks lymphangiogenesis in vivo (FIG. 5 m).

Example 8 Naphthyridine Inhibits VEGFA-Induced (Lymph)Angiogenesis inMice

The adenosine A1 receptor antagonist naphthyridine was identified as anovel inhibitor of lymphatic and blood vessel formation in Xenopustadpoles and in vitro cellular assays, where it acted anti-mitogenic andimpaired endothelial tube formation. It was determined that naphtyridineinhibited LEC tube formation with an IC₅₀ of 1.3 μM±0.1 μM (data notshown). To investigate whether naphthyridine might also inhibitmammalian angiogenesis and/or lymphangiogenesis in vivo,VEGFA-containing Matrigel plugs were implanted subcutaneously into adultmice and treated these mice systemically with naphthyridine (3mg/kg/day) or with vehicle control for 6 days. In agreement withprevious results (Hong et al., 2004), differential immunofluorescenceanalysis for the lymphatic-specific marker LYVE1 and the panvascularmarker CD31 demonstrated lymphatic vessel enlargement in the skinsurrounding VEGFA-containing Matrigels, as compared with controlMatrigels only containing PBS (FIG. 8 a, b). Treatment withnaphthyridine resulted in a reduction of blood vessel numbers andlymphatic vessel enlargement (FIG. 8 c). The quantitative image analysesshown in FIG. 8 d-g confirmed that the tissue area covered by lymphaticvessels surrounding VEGFA containing Matrigels was significantly reducedby treatment with naphthyridine in comparison to the PBS control(1.36±0.36% versus 0.89±0.18%; p=0.05; FIG. 8 e), while the density oflymphatic vessels was unchanged by VEGFA or naphthyridine treatment(FIG. 8 d). Treatment with naphthyridine also reduced the number ofVEGFA-induced blood vessels (36.49±1.49 versus 47.93±3.55; p=0.0021;FIG. 8 f) and resulted in a reduction of the tissue area covered byblood vessels (FIG. 8 g). It can be concluded that the ability ofnaphthyridine to inhibit lymph and blood vessel angiogenesis isconserved between Xenopus and mammals.

All of the above-cited references and publications are incorporated byreference herein in their entireties.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific method and reagents described herein. Such equivalents areconsidered to be within the scope of this invention and are covered bythe following claims.

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TABLE 1 Summary of the results from the phenotypic chemical libraryscreen of Xenopus embryos Number of % of compounds Phenotype compoundstested Normal embryogenesis 1166 91.1 Abnormal embryogenesis 114 8.9Total 1280 100.0 Phenotype classes Cytotoxicity 32 2.5 Lethality 18 1.4Edema formation 48 3.7 Pigmentation defects 10 0.8 Other defects 6 0.5Total 114 8.9

TABLE 2 Compilation of 32 compounds causing cytotoxicity in Xenopusembryos Lethality Name Secondary Name Class Enzyme Action Selectivity(NF) Niclosamide 2′,5′-Dichloro-4′- Antibiotic Protonophore 32nitrosalicylanilide beta-Lapachone Apoptosis Activator 32 Z-L-Phechloromethyl N-Carbobenzyloxy-L- Biochemistry Enzyme InhibitorChymotrypsin A- 32 ketone phenylalanyl chloromethyl gamma ketone; ZPCK(R)-(+)-WIN 55,212-2 (R)-(+)-[2,3-Dihydro-5- Cannabinoid Agonist 32mesylate methyl- 3[(morpholinyl)methyl]pyrrolo[1,2,3- de]-1,4-benzoxazinyl]-(1- naphthalenyl)methanone mesylate Bay 11-7085(E)-3-(4-t- Cell Cycle Inhibitor IkB-alpha 32 Butylphenylsulfonyl)-2-propenenitrile Rotenone Cell Stress Modulator Mitochondria 32 2-(alpha-alpha-NETA Cholinergic Enzyme Inhibitor Choline 32Naphthoyl)ethyltrimethylammonium Acetyltransferase iodide 5-Nitro-2-(3-NPPB Cl-Channel Blocker 32 phenylpropylamino)benzoic acid Brefeldin Afrom BFA; Ascotoxin, Cyanein Cytoskeleton and Inhibitor Golgi apparatus33/34 Penicillium ECM brefeldianum Fluspirilene R 6218 DopamineAntagonist D2/D1 32 Farnesylthiosalicylic FTS G protein EnzymeAntagonist Ras 32 acid Calcimycin A23187; Calcium Intracellular Ca2+ 32ionophore A23187 Calcium Thapsigargin Intracellular Enzyme Releaser 32Calcium Sanguinarine chloride 13-Methyl- Ion Pump Inhibitor Na+/K+ATPase 32 [1,3]benzodioxolo[5,6-c]- 1,3-dioxolo[4,5-i]phenanthridiniumchloride Ebselen 2-Phenyl-1,2- Leukotriene Enzyme InhibitorLipoxygenases/ 32 benzisoselenazol-3(2H)- glutathione S- one transferaseSR 2640 2-[[3-(2- Leukotriene Antagonist CysLT1 32Quinolinylmethoxy)phenyl]amino]- benzoic acid; QMPB D-609 potassiumCarbonodithioic acid, O- Lipid Enzyme Inhibitor PIPLC 33/34(octahydro-4,7-methano- 1H-inden-5-yl) ester potassium MJ331-Hexadecyl-3- Lipid Enzyme Inhibitor PLA2 32 (trifluoroethyl)-sn-glycero-2- phosphomethanol lithium Ro 41-0960 2′-Fluoro-3,4-dihydroxy-5-Neurotransmission Enzyme Inhibitor COMT 33/34 nitrobenzophenoneCantharidic Acid Phosphorylation Enzyme Inhibitor PP1/PP2A 32Cantharidin Cantharidine Phosphorylation Enzyme Inhibitor PP2A 32Dequalinium analog, C14 Linker; DECA-14; Phosphorylation EnzymeInhibitor PKC-alpha 32 C-14 linker Quinolinium Palmitoyl-DL-Phosphorylation Enzyme Modulator PKC 32 Carnitine chloride Phorbol12-myristate PMA Phosphorylation Enzyme Activator PKC 32 13-acetaterac-2-Ethoxy-3- Phosphorylation Enzyme Inhibitor PKC 32hexadecanamido-1- propylphosphocholine rac-2-Ethoxy-3- PhosphorylationEnzyme Inhibitor PKC 32 octadecanamido-1- propylphosphocholine RottlerinMallotoxin Phosphorylation Enzyme Inhibitor PKC/CaM 32 Kinase IIITyrphostin A9 [[3,5-bis(1,1- Phosphorylation Enzyme Inhibitor PDGFR 32Dimethylethyl)-4- hydroxyphenyl]methylene]- propanedinitrile TyrphostinAG 879 alpha-cyano-(3,5-di-t- Phosphorylation Enzyme Inhibitor TrkA 32butyl-4- hydroxy)thiocinnamide Wortmannin from Phosphorylation EnzymeInhibitor PI3K 33/34 Penicillium funiculosum L-655,2403-[1-(4-Chlorobenzyl)-5- Thromboxane Antagonist TXA2 32fluoro-3-methyl-indol-2- yl]-2,2-dimethyl propanoic acid GW76472-(4-(2-(1- Transcription Agonist PPAR-alpha 32 Cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)- 2- methylpropionic acid

TABLE 3 Compilation of 18 compounds causing lethality in Xenopus embryosLethality Name Secondary Name Class Enzyme Action Selectivity (NF) FPL64176 2,5-Dimethyl-4-[2- Ca2+ Channel Activator L-type 40(phenylmethyl)benzoyl]- 1H-pyrrole-3- carboxylic acid methyl esterNitrendipine 1,4-Dihydro-2,6- Ca2+ Channel Antagonist L-type 45dimethyl-4-(3- nitrophenyl)-3,5- pyridinecarboxylic acid ethyl methylester TG003 (Z)-1-(3-Ethyl-5- Cell Cycle Enzyme Inhibitor Clk 40methoxy-2,3- dihydrobenzothiazol-2- ylidene)-propan-2-one IvermectinMK-933 Cholinergic Modulator alpha7 nACh 41/42 Loratadine4-(8-chloro-5,6-dihydro- Histamine Antagonist H1 45 11H-benzo[5,6]cycloheptal[1,2- b]pyridin-11-ylidene- 1-piperidinecarboxylicacid ethyl ester (R,R)-cis-Diethyl (5R,11R)-5,11-Diethyl- HormoneAntagonist ER-beta 41 tetrahydro-2,8- 5,6,11,12-tetrahydro- chrysenediol2,8-chrysenediol Calmidazolium R 24571 chloride Intracellular CalciumEnzyme Inhibitor Ca2+ATPase 37/38 chloride MK-8863[3-tert-Butylthio-1-(4- Leukotriene Inhibitor 39 chlorobenzyl)-5-isopropyl-1H-indol-2- yl]-2,2- dimethylpropionic acid, sodium saltET-18-OCH3 3,5,9-Trioxa-4- Lipid Enzyme Inhibitor PIPLC 37/38phosphaheptacosan-1- aminium L-162,313 (5,7-dimethyl-2-ethyl-3-Neurotransmission Agonist AT1 45 [[4-[2(n- butyloxycarbonylsulfonamido)-5-isobutyl-3- thienyl]phenyl]- methyl]imidazo[4,5,6]pyridineN-Oleoyldopamine OLDA Neurotransmission Ligand CB1 41DL-Stearoylcarnitine Phosphorylation Enzyme Inhibitor PKC 41 chlorideIndirubin-3′-oxime Indirubin-3′-monoxime Phosphorylation EnzymeInhibitor CDK 45 NSC 95397 2,3-bis[(2- Phosphorylation Enzyme InhibitorCdc25 40 Hydroxyethyl)thio]-1,4- naphthoquinone Purvalanol A NG-60Phosphorylation Enzyme Inhibitor CDK 41/42 SP600125 Anthrapyrazolone;1,9- Phosphorylation Enzyme Inhibitor c-JNK 41 Pyrazoloanthrone SU 54161,3-Dihydro-3-[(3,5- Phosphorylation Inhibitor VEGFR PTK 37/38dimethyl-1H-pyrrol-2-yl)methylene]- 2H-indol-2- one (±)-Ibuprofenalpha-Methyl-4- Prostaglandin Enzyme Inhibitor COX 45(isobutyl)phenylacetic acid

TABLE 4 Compilation of 48 compounds causing edema in Xenopus embryosOnset Lethality Name Secondary Name Class Enzyme Action Selectivity (NF)(NF) 1,3-Diethyl-8- DPX Adenosine Antagonist A1 41/42 45 phenylxanthine7-Chloro-4- Adenosine Antagonist A1 39 39 hydroxy-2-phenyl-1,8-naphthyridine CGS-15943 9-Chloro-2-(2- Adenosine Antagonist A1 41/4246 furyl)[1,2,4]triazolo[1,5- c]quinazolin-5-amine L-765,314(2S)-4-(4-Amino-6,7- Adrenoceptor Antagonist alpha-1B 41/42 —dimethoxy-2- quinazolinyl)-2-[[(1,1- Dimethylethyl)- amino]carbonyl]- 1-piperazinecarboxylic acid, phenylmethyl ester Mevastatin CompactinAntibiotic Enzyme Inhibitor Ras, Rho 35/36 45 4-Amino-1,8- ApoptosisEnzyme Inhibitor PARP 41/42 — naphthalimide Retinoic acid Vitamin A acidApoptosis Enzyme Activator 39 40 1,10- o-Phenanthroline BiochemistryEnzyme Inhibitor Metalloprotease 39/40 46 Phenanthroline monohydratemonohydrate Felodipine Plendil Ca2+ Channel Blocker L-type 41/42 45 MRS1845 N-Propargylnitrendipene Ca2+ Channel Inhibitor SOC 41/42 45Nicardipine YC-93 hydrochloride Ca2+ Channel Antagonist L-type 41/42 45hydrochloride Nifedipine Ca2+ Channel Antagonist L-type 41/42 46Nimodipine 1,4-Dihydro-2,6-dimethyl- Ca2+ Channel Antagonist L-type41/42 41/42 4-(3-nitrophenyl)- 3,5-pyridinecarboxylic acid2-methoxyethyl 1- methylethyl ester SKF 96365 1-(beta-[3-(4- Ca2+Channel Inhibitor 39 41/42 Methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride (±)-alpha-Lipoic(±)-1,2-Dithiolane-3- Cell Stress Enzyme Coenzyme Pyruvate 39 45 Acidpentanoic acid dehydrogenase Forskolin Cyclic Enzyme Activator Adenylate39/40 41/42 Nucleotides cyclase YC-1 3-(5′-Hydroxymethyl-2′- CyclicEnzyme Activator Guanylyl 45 — furyl)-1-benzyl indazole Nucleotidescyclase Zardaverine 6-(4-Difluoromethoxy-3- Cyclic Enzyme Inhibitor PDEIII/PDE IV 39/40 43 methoxyphenyl)-3(2H)- Nucleotides pyridazinoneNocodazole R 17934 Cytoskeleton Inhibitor beta-tubulin 33/34 33/34 andECM Podophyllotoxin Cytoskeleton Inhibitor 39/40 41/42 and ECM GBR-129091-[2-[bis(4- Dopamine Inhibitor Reuptake 45 — dihydrochlorideFluorophenyl)methoxy]ethyl]- 4-[3- phenylpropyl]piperazinedihydrochloride N-(p- NIPS hydrochloride Dopamine Antagonist D2 45 —Isothiocyanatophenethyl)- spiperone hydrochloride Pimozide DopamineAntagonist D2 41/42 45 Sobuzoxane 4,4′-(1,2- Gene Enzyme Inhibitor TopoII 41/42 47 Ethanediyl)bis(1- Regulation isobutoxycarbonyloxymethyl-2,6-piperazinedione) Riluzole 2-Amino-6- Glutamate Antagonist Release 45— (trifluoromethoxy)- benzothiazole Clemastine Histamine Antagonist H145 — fumarate 2-methoxyestradiol 2-Hydroxyestradiol 2- HormoneMetabolite Estrogen 33/34 33/34 methyl ether beta-EstradiolDihydrofolliculin Hormone Estrogen 42 45 Dequalinium1,1′-Decamethylenebis(4- K+ Channel Blocker 39 39 dichlorideaminoquinaldinium)dichloride 7-Cyclopentyl-5-(4- Phosphorylation EnzymeInhibitor Ick 41/42 45 phenoxy)phenyl- 7H-pyrrolo[2,3- d]pyrimidin-4-ylamine Cyclosporin A Antibiotic S 7481F1 Phosphorylation EnzymeInhibitor Calcineurin 41/42 — phosphatase Diacylglycerol R 59022Phosphorylation Enzyme Inhibitor Diacylglycerol 45 — kinase inhibitor Ikinase Diacylglycerol R59949 Phosphorylation Enzyme InhibitorDiacylglycerol 41/42 — Kinase Inhibitor II kinase Genistein5,7-Dihydroxy-3-(4- Phosphorylation Enzyme Inhibitor Tyrosine kinase35/36 46 hydroxyphenyl)-4H-1- benzopyran-4-one GW2974N4-(1-Benzyl-1H-indazol- Phosphorylation Enzyme Inhibitor EGFR/ErbB-241/42 47 5-yl)-N6,N6-dimethyl- pyrido[3,4-d]pyrimidine4- 4,6-diamineGW5074 3-(3,5-Dibromo-4- Phosphorylation Enzyme Inhibitor Raf1 kinase33/34 41/42 hydroxybenzylidine-5- iodo-1,3-dihydro-indol-2- one) IC 2611,3-Dihydro-3-[(2,4,6- Phosphorylation Enzyme Inhibitor CK- 35/36 41/42trimethoxyphenyl)methylene]- 1delta/epsilon 2H-indol-2-one KenpaulloneNSC 664704 Phosphorylation Enzyme Inhibitor CDK1, CDK2, 45 — CDK5 SU4312 3-(4- Phosphorylation Enzyme Inhibitor KDR 45 —Dimethylaminobenzylidenyl)- 2-indolinone SU 6656 2,3-Dihydro-N,N-Phosphorylation Enzyme Inhibitor Src family 39/40 46 dimethyl-2-oxo-3-kinase [(4,5,6,7-tetrahydro-1H- indol-2-yl)methylene]-1H-indole-5-sulfonamide Tyrphostin AG N-(3-Chlorophenyl)-6,7-Phosphorylation Enzyme Inhibitor EGFR 45 46 1478 dimethoxy-4-quinazolinamine Tyrphostin AG 494 N-Phenyl-3,4- Phosphorylation EnzymeInhibitor EGFR 39 40 dihydroxy- benzylidenecyanoacetamide Meclofenamicacid 2-([2,6-Dichloro-3- Prostaglandin Enzyme Inhibitor COX/5- 35/3641/42 sodium methylphenyl]amino)benzoic Lipoxygenase acid sodiumAmperozide 4-[4,4-bis(4- Serotonin Ligand 41/42 — hydrochlorideFluorophenyl)butyl]-N- ethyl-1- piperazinecarboxamide hydrochlorideRitanserin 6-[2-[4-bis(4- Serotonin Antagonist 5-HT2/5-HT1C 41/42 —Fluorophenyl)methylene]- 1-piperidinyl]- ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin- 5-one 13-cis-retinoic acid IsotretinoinTranscription Regulator RAR-alpha, 41/42 46 beta 6(5H)- TranscriptionEnzyme Inhibitor PARP 45 47 Phenanthridinone TTNPB Arotinoid acidTranscription Ligand RAR-alpha, 39 45 beta, gamma

TABLE 5 Compilation of 10 compounds causing pigmentation defects inXenopus embryos. Onset Lethality Name Secondary Name Class Enzyme ActionSelectivity Phenotypes (NF) (NF) (−)-Ephedrine Adrenoceptor ActivatorLack of skin 33/34 41/42 hemisulfate pigmentation Nalidixic acid1-Ethyl-1,4- Antibiotic Enzyme Inhibitor DNA Gyrase Silvery-colored 47 —sodium dihydro-7-methyl- embryos 4-oxo-1,8- naphthyridine-3- carboxylicacid sodium Caffeic acid CAPE Cell Cycle Inhibitor NFkB Lack of skin33/34 39 phenethyl pigmentation ester 1-Phenyl-3-(2- Dopamine EnzymeInhibitor beta- Reduced eye & 33/34 — thiazolyl)-2- Hydroxylase skinpigmentatiion thiourea Amfonelic 7-Benzyl-1-ethyl- Dopamine ModulatorReduced eye & 33/34 — acid 1,4-dihydro-4-oxo- skin pigmentatiion1,8-naphthyridine- 3-carboxylic acid Apomorphine 10,11- Dopamine AgonistReduced eye & 33/34 — hydrochloride Dihydroxyaporphine skinpigmentatiion hemihydrate hydrochloride hemihydrate R(−)-N- DopamineAgonist Lack of skin 33/34 40 Allylnorapomorphine pigmentation;hydrobromide Delayed embryogenesis; Paralyzed embryos R(−)- R(−)-NPADopamine Agonist D2 Reduced skin 33/34 — Propylnorapomorphinehydrochloride pigmentatiion hydrochloride Spiroxatrine R 5188 SerotoninAgonist 5-HT1A Increased skin 45 — pigmentation WIN 62,57717-beta-Hydroxy- Tachykinin Antagonist NK1 Reduced eye & 33/34 4217-alpha-ethynyl- skin pigmentatiion delta-4- androstano(3,2-b)pyrimido(1,2- a)benzimidazole

TABLE 6 Compilation of 6 compounds causing other phenotypic defects inXenopus embryos. Onset Lethality Name Secondary Name Class Enzyme ActionSelectivity Phenotypes (NF) (NF) (S)-(+)- Apoptosis Enzyme InhibitorTopol Worm-like, crippled 39 41/42 Camptothecin embryos; Tremoringembryos; Very small eyes 2,3- DMNQ Cell Stress Modulator Browndiscoloration; 41 42 Dimethoxy- Tremoring embryos 1,4- naphthoquinone SB205384 4-Amino-7- GABA Modulator GABA-A Brown discoloration; 40 46hydroxy-2-methyl- Delayed 5,6,7,8- embryogenesis tetrahydrobenzo-[b]thieno[2,3- b]pyridine- 3-carboxylic acid but-2-ynyl ester trans-5-Androsten-3beta- Hormone Aldosterone Brown discoloration; 41/42 —Dehydroandrosterone ol-17-one; DHEA Tremoring embryos 3- Multi-DrugEnzyme Substrate CYP450 Swollen vacuoles in 46 — AminopropionitrileResistance the tail's notochord fumarate Diphenyleneiodonium[1,1′-Biphenyl]-2,2′- Nitric Oxide Enzyme Inhibitor eNOS Browndiscoloration; 41 42 chloride diyiodonium Tremoring embryos chloride

TABLE 7 Bioactive pharmacological classes by phenotype ScreenCytotoxicity Lethality Edema Pigmentation Other Total Total Active %Active % Active % Active % Active % Class⁺ agents active agents totalagents total agents total agents total agents total Adenosine 55 3 3 5.5Adrenoreceptor 104 2 1 1.0 1 1.0 Antibiotic 28 3 1 3.6 1 3.6 1 3.6Apoptosis 12 4 1 8.3 2 16.7 1 8.3 Biochemistry 46 2 1 2.2 1 2.2 Ca²⁺Channel 18 8 2 11.1 6 33.3 Cannabinoid 6 1 1 16.7 Cell cycle 15 3 1 6.71 6.7 1 6.7 Cell stress 19 3 1 5.3 1 5.3 1 5.3 Cholinergic 77 2 1 1.3 11.3 Cl⁻ Channel 3 1 1 33.3 Cyclic nucleotides 31 3 3 9.7 Cytoskeleton &10 3 1 10.0 2 20.0 ECM Dopamine 114 9 1 0.9 3 2.6 5 4.4 G protein 4 1 125.0 GABA 42 1 1 2.4 Gene regulation 1 1 1 100.0 Glutamate 88 1 1 1.1Histamine 31 2 1 3.2 1 3.2 Hormone 34 4 1 2.9 2 5.9 1 2.9 Intracellular8 3 2 25.0 1 12.5 calcium Ion pump 17 1 1 5.9 K⁺ Channel 19 1 1 5.3Leukotriene 10 3 2 20.0 1 10.0 Lipid 10 3 2 20.0 1 10.0 Multi-drug 12 11 8.3 resistance Neurotransmission 46 3 1 2.2 2 4.3 Nitric oxide 37 1 12.7 Phosphorylation 92 30 11 12.0 6 5.5 13 14.1 Prostaglandin 24 2 1 4.21 4.2 Serotonin 87 3 2 2.3 1 1.1 Tachykinin 5 1 1 20 Thromboxane 2 1 150.0 Transcription 12 4 1 8.3 3 25.0 Entire Screen 1280 114 32 2.5* 181.4* 48 3.8* 10 0.8* 6 0.5* ⁺Includes only bioactive classes. *Frequencyof whole screen.

TABLE 8 Phenotypic classification of edema-inducing compounds Type ofedema Phenotype Tail Cardiac Lymph heart class Compound namePharmacological class Selectivity Cerebral Periocular PericardialVentral Proctodeal Pronephric tip phenotype enlargement Lethality A1,3-Diethyl-8- Adenosine A1 41/42 41/42 45 phenylxanthine CGS-15943Adenosine A1 41/42 45 45 46 Mevastatin Antibiotic Ras, Rho 35/36 41/42 §45 1,10-Phenanthroline Biochemistry Metallo- 39/40 41/42 46 monohydrateprotease Felodipine Ca2+ Channel L-type 41/42 41/42 41/42 45 MRS 1845Ca2+ Channel SOC 41/42 41/42 41/42 41/42 45 Nicardipine Ca2+ ChannelL-type 41/42 41/42 45 hydrochloride Nifedipine Ca2+ Channel L-type 41/4241/42 45 46 Nimodipine Ca2+ Channel L-type 41/42 41/42‡ 41/42¶ 41/42 SKF96365 Ca2+ Channel 39 40 40 41/42 Forskolin Cyclic Adenylate 39 39 3941/42 Nucleotides cyclase Pimozide Dopamine D2 41/42 41/42 41/42 41/42¶45 Clemastine fumarate Histamine H1 46 46 45 — GW2974 PhosphorylationEGFR/ 41/42 41/42 41/42 45 47 ErbB2 Amperozide Serotonin 45 41/42 41/42— hydrochloride 6(5H)-Phenanthridinone Transcription PARP 45 45 45 47 BL-765,314 Adrenoceptor alpha-1B 41/42# 45 — YC-1 Cyclic Guanylyl 45 45 —Nucleotides cyclase GBR-12909 Dopamine Reuptake 45 45 45 —dihydrochloride N-(p- Dopamine D2 45 45 —Isothiocyanatophenethyl)spiperone hydrochloride Riluzole GlutamateRelease 45 45 45 — beta-Estradiol Hormone Estrogen 42# 45 Cyclosporin APhosphorylation Calcineurin 45 45 41/42 — phosphatase Diacylglycerolkinase Phosphorylation Diacyglycerol 45 45 — inhibitor I kinaseDiacylglycerol Kinase Phosphorylation Diacyglycerol 46 41/42 45 —Inhibitor II kinase Kenpaullone Phosphorylation CDK1, CDK2, 45 45 — CDK5SU 4312 Phosphorylation KDR 45 45 45 — Ritanserin Serotonin 5-HT2/5-41/42 41/42 45 — HT1C C Naphthyridine Adenosine A1 39 39(±)-alpha-Lipoic Acid Cell stress Pyruvate de- 39 42 45 hydrogenaseZardaverine Cyclic PDE III/PDE 39/40 41/42 43 Nucleotides IV NocodazoleCytoskeleton and beta-tubulin 33/34 33/34 ECM PodophyllotoxinCytoskeleton and 39/40 41/42 ECM 2-methoxyestradiol Hormone Estrogen33/34 33/34 Dequalinium dichloride K+ Channel 39 39 7-CycloPhosphorylation Ick 41/42 41/42 45 GW5074 Phosphorylation Raf1 kinase33/34 41/42 Meclofenamic acid Prostaglandin COX/5- 35/36 41/42 sodiumLipoxygenase D 4-Amino-1,8- Apoptosis PARP 41/42* 41/42 47 —naphthalimide Sobuzoxane Gene Regulation Topo II 45* 45 45 41/42 47 SU6656 Phosphorylation Src family 41/42* 41/42 39/40 39/40 39/40 46kinases E Retinoic acid Apoptosis 39+ 40 39 39 13-cis-retinoic acidTranscription RAR-alpha, 45+ 41/42 45 46 beta TTNPB TranscriptionRAR-alpha, 41/42+ 41/42 41/42 39 45 beta, gamma F GenisteinPhosphorylation Tyrosine 35/36 41/42 kinase IC 261 Phosphorylation CK-135/36 41/42 41/42 § 41/42 delta/epsilon Tyrphostin AG 1478Phosphorylation EGFR 45 45 45¶ 46 Tyrphostin AG 494 Phosphorylation EGFR39 § 40 *Periocular edema causing bulging eyes. #Bilateral periocularedemas causing narrow-set eyes. +Cerebral edema extendingdorso-anteriorly and displacing the eyes. § Heart tube failed to loop.¶The heart chambers were enlarged. ‡The edemas were also dectected inthe gut. Numbers shown represent embryonic stages according to Nieuwkoopand Faber (NF). Compound abbreviations: 7-Cyclo,7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine;Naphthyridine, 7-Chloro-4-hydroxy-2-phenyl-1,8-naphthyridine.

TABLE 9 Vascular phenotypes resulting from small-molecule treatment ofXenopus embryos A) Compounds affecting blood vessel development onlyPhenotype Compound Pharmacological ISV VVN PCV Embr. class name classSelectivity phenotype phenotype phenotype Lymphatic phenotype phen.Defective Nocodazole Cytoskeleton beta- impaired hypoplastic hypoplasticn.a. EDE vasculogenesis and ECM tubulin Podophyllotoxin Cytoskeletonimpaired hypoplastic hypoplastic n.a. EDE and ECM 2-methoxyestradiolHormone Estrogen impaired hypoplastic hypoplastic n.a. EDE IC 261Phosphorylation CK- impaired hypoplastic hypoplastic n.a. EDE1delta/epsilon NSC 95397 Phosphorylation Cdc25 impaired hypoplastichypoplastic n.a. LET Tyrphostin AG Phosphorylation EGFR impairedhypoplastic hypoplastic n.a. EDE 494 Meclofenamic Prostaglandin COX/5-impaired hypoplastic hypoplastic n.a. EDE acid sodium LipoxygenaseDefective Retinoic acid Apoptosis impaired hypoplastic normal n.a. EDEangiogenesis Forskolin Cyclic Adenylate impaired hypoplastic normal n.a.EDE nucleotides cyclase L-162,313 Neurotransmission AT1 impairedhypoplastic normal normal LET Indirubin-3′- Phosphorylation CDK impairednormal normal normal LET oxime SU 5416 Phosphorylation VEGFR impairedhypoplastic normal n.a. LET PTK Ectopic Calmidazolium IntracellularCa2+ATPase ectopic hyperplastic normal n.a. LET angiogenic chlorideCalcium ISVs sprouting GW5074 Phosphorylation Raf1 ectopic dysplasticnormal n.a. EDE kinase ISVs VVN YC-1 Cyclic Guanylyl normal hypoplasticnormal normal EDE hypoplasia nucleotides cyclase Zardaverine Cyclic PDEnormal hypoplastic normal n.a. EDE nucleotides III/PDE IV MK-886Leukotriene normal hypoplastic normal n.a. LET Purvalanol APhosphorylation CDK normal hypoplastic normal normal LET B) Compoundsaffecting blood and lymph vessel formation Phenotype CompoundPharmacological ISV VVN PCV ALS ALH PLV Embr. class name classSelectivity phenotype phenotype phenotype phenotype phenotype phenotypephen. Defective Naphthyridine Adenosine A1 stunted hypoplastic normalstunted dysplastic hypoplastic EDE blood and 7-Cyclo Phosphorylation Ickstunted normal normal impaired impaired hypoplastic EDE lymph SP600125Phosphorylation c-JNK stunted dysplastic normal impaired impairedimpaired LET angiogenesis VVN 1,3-Diethyl- Adenosine A1 normalhyperplastic normal stunted stunted hypoplastic EDE hyperplasia8-phenylxanthine and 4-Amino-1,8- Apoptosis PARP normal hyperplasticnormal impaired stunted hypoplastic EDE defective naphthalimide lymphGenistein Phosphorylation Tyrosine stunted hyperplastic normal impairedimpaired impaired EDE angiogenesis kinase C) Compounds affecting lymphvessel formation only Pharmacological ALH Embr. Phenotype class Compoundname class Selectivity Blood vessel phenotype ALS phenotype phenotypePLV phenotype phen. Defective Felodipine Ca2+ L-type normal normaldysplastic normal EDE lymph Channel angiogenesis Sobuzoxane Gene Topo IInormal dysplastic dysplastic hypoplastic EDE regulation Nicardipine Ca2+L-type normal dysplastic dysplastic hypoplastic EDE hydrochlorideChannel SU 6656 Phosphorylation Src family normal stunted dysplastichypoplastic EDE kinase Dequalinium K+ channel normal stunted stuntedhypoplastic EDE dichloride GW2974 Phosphorylation EGFR/ normal impairedstunted hypoplastic EDE ErbB2-2 Mevastatin Antibiotic Ras, Rho normalimpaired impaired hypoplastic EDE SU 4312 Phosphorylation KDR normalimpaired impaired hypoplastic EDE Abbreviations: ALH, anterior lymphheart; ALS, anterior lymph sac; EDE, edema phenotype; ISV, intersomiticvessels; LET, lethal before stage 48; n.a., not applicable; PCV,posterior cardinal vein; Phen, phenotype; PLV, posterior lymph vessels;VVN, vitelline vein network. Compound abbreviations: 7-Cyclo,7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine;Naphthyridine, 7-Chloro-4-hydroxy-2-phenyl-1,8-naphthyridine.

TABLE 10 Comparison between the in vivo and in vitro activities ofsmall-molecule compounds Phenotype class in HUVEC HUVEC tube LEC LECtube vivo Compound name proliferation formation proliferation formationA) Compounds affecting in vivo blood vessel development only DefectiveNocodazole − − − − − − vasculogenesis Podophyllotoxin − − − − − −2-methoxyestradiol ◯ − − − − IC 261 ◯ − − − Tyrphostin AG 494 ◯ ◯ ◯ −Defective angiogenesis Retinoic acid + − + − L-162,313 + − ◯ −Indirubin-3′-oxime − − ◯ ◯ SU 5416 ◯ ◯ ◯ − Ectopic angiogenic GW5074 ◯◯ + − sprouting VVN hypoplasia MK-886 + − ◯ − B) Compounds affecting invivo blood and lymph vessel formation Defective blood and Naphthyridine− − − − − − lymphangiogenesis 7-Cyclo ◯ − − ◯ − − SP600125 ◯ ◯ ◯ − VVNhyperplasia and 1,3-Diethyl-8- ◯ + ◯ ◯ defective phenylxanthinelymphangiogenesis 4-Amino-1,8-naphthalimide + ◯ ◯ − Genistein ◯ − ◯ − C)Compounds affecting in vivo lymph vessel formation only DefectiveFelodipine ◯ − ◯ ◯ lymphangiogenesis Sobuzoxane + ◯ + ◯ Nicardipinehydrochloride ◯ ◯ ◯ ◯ GW2974 + − + − SU 4312 + ◯ ◯ ◯ D) Compoundswithout vascular defects in vivo — Cyclosporin A ◯ ◯ ◯ ◯ MRS 1845 + ◯ ◯− L-687,384 hydrochloride + − ◯ − Scoring system for in vitro assays: ◯,inactive or marginal effects (85-115% of control); −, moderateinhibition (50-85% of control); − −, strong inhibition (<50% ofcontrol); +, moderate promotion (>115-150% of control). Abbreviations:HUVEC, human umbilical vein endothelial cell; LEC, lymphatic endothelialcell; VVN, vitelline vein network. Compound abbreviations: 7-Cyclo,7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine;Naphthyridine, 7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine.

1. A method for in vivo screening comprising: a) treating a plurality ofamphibians with a plurality of agents; b) identifying an amphibian fromthe plurality of amphibians wherein the treatment causes edema in ordeath of the amphibian; and c) determining the anatomical pattern ofedema formation in the identified amphibian.
 2. The method of claim 1,wherein the plurality of amphibians are treated by including theplurality of agents in the culture media containing the plurality ofamphibians.
 3. The method of claim 2, wherein the plurality of agentsare dissolved in the culture media containing the plurality ofamphibians.
 4. The method of claim 1, wherein the method is performed ina multi-well format.
 5. The method of claim 1, wherein the plurality ofamphibians are a plurality of embryos, tadpoles, or adults.
 6. Themethod of claim 1, wherein the plurality of amphibians are from thesubclass Lissamphibia.
 7. The method of claim 6, wherein the pluralityof amphibians are frogs, toads, newts, salamanders, mudpuppies, orcaecilians.
 8. The method of claim 1, wherein the plurality ofamphibians are from the genus Xenopus.
 9. The method of claim 8, whereinthe plurality of amphibians are from the species Xenopus laevis orXenopus tropicalis.
 10. The method of claim 1, wherein the plurality ofagents are independently small molecules, drugs, antibodies, peptides,secreted proteins, nucleic acids, antisense RNA molecules, ribozymes,RNA interference nucleotide sequences, antisense oligomers, ormorpholino oligonucleotides.
 11. The method of claim 1, wherein theedema or death is caused by an activity in the vascular, lymphatic,cardiac, or excretory system of the identified amphibian.
 12. The methodof claim 1, further comprising the step of identifying the target tissueor organ of the agent responsible for the edema or death in theidentified amphibian.
 13. The method of claim 1, wherein the anatomicalpattern of edema formation in the identified amphibian is cerebral,periocular, pericardial, ventral, proctodeal, pronephric, or tail tip.14. The method of claim 1, wherein the anatomical pattern of edemaformation in the identified amphibian is a cardiac phenotype or alymph-heart enlargement.
 15. The method of claim 1, wherein the methodfurther comprises a secondary screen.
 16. The method of claim 15,wherein the secondary screen is performed by in situ hybridization. 17.The method of claim 16, wherein the in situ hybridization is performedmanually, semi-automated or fully automated.
 18. The method of claim 15,wherein the secondary screen is performed by immunohistochemistry. 19.The method of claim 18, wherein the immunohistochemistry is performedmanually, semi-automated or fully automated.
 20. The method of claim 1,wherein the method further comprises the step of d) identifying theagent causing the edema in or death of the amphibian. 21-49. (canceled)50. The method of claim 1, wherein the method further comprises the stepof d) identifying a pathway that mediates lymphatic and/or vasculardevelopment in the identified amphibian.
 51. The method of claim 50,wherein the pathway is a VEGF pathway.
 52. The method of claim 51,wherein the pathway is targeted by an adenosine receptor antagonist.